Nucleic acid compositions for stimulating immune responses

ABSTRACT

The invention provides an immunostimulatory nucleic acid comprising CpG motifs, and methods of use thereof in stimulating immunity.

RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Serial No. 60/394,164, entitled “NUCLEIC ACIDCOMPOSITIONS FOR STIMULATING IMMUNE RESPONSES”, filed on Jul. 3, 2002,which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to immunostimulatorynucleic acids, compositions thereof and methods of using theimmunostimulatory nucleic acids.

BACKGROUND OF THE INVENTION

[0003] Bacterial DNA has immune stimulatory effects to activate B cellsand natural killer cells, but vertebrate DNA does not (Tokunaga, T., etal., 1988. Jpn. J. Cancer Res. 79:682-686; Tokunaga, T., et al., 1984,JNCI 72:955-962; Messina, J. P., et al., 1991, J. Immunol.147:1759-1764; and reviewed in Krieg, 1998, In: Applied OligonucleotideTechnology, C. A. Stein and A. M. Krieg, (Eds.), John Wiley and Sons,Inc., New York, N.Y., pp. 431-448). It is now understood that theseimmune stimulatory effects of bacterial DNA are a result of the presenceof unmethylated CpG dinucleotides in particular base contexts (CpGmotifs), which are common in bacterial DNA, but methylated andunderrepresented in vertebrate DNA (Krieg et al, 1995 Nature374:546-549; Krieg, 1999 Biochim. Biophys. Acta 93321:1-10).

[0004] The immune stimulatory effects of bacterial DNA can be mimickedwith synthetic oligodeoxynucleotides (ODN) containing these CpG motifs.Such CpG ODN have highly stimulatory effects on human and murineleukocytes, inducing B cell proliferation; cytokine and immunoglobulinsecretion; natural killer (NK) cell lytic activity and IFN-γ secretion;and activation of dendritic cells (DCs) and other antigen presentingcells to express costimulatory molecules and secrete cytokines,especially the Th1-like cytokines that are important in promoting thedevelopment of Th1-like T cell responses. These immune stimulatoryeffects of native phosphodiester backbone CpG ODN are highly CpGspecific in that the effects are essentially abolished if the CpG motifis methylated, changed to a GpC, or otherwise eliminated or altered(Krieg et al, 1995 Nature 374:546-549; Hartmann et al, 1999 Proc. Natl.Acad. Sci USA 96:9305-10). Phosphodiester CpG ODN can be formulated inlipids, alum, or other types of vehicles with depot properties orimproved cell uptake in order to enhance the immune stimulatory effects(Yamamoto et al, 1994 Microbiol. Immunol. 38:831-836; Gramzinski et al,1998 Mol. Med. 4:109-118).

[0005] In early studies, it was thought that the immune stimulatory CpGmotif followed the formula purine-purine-CpG-pyrimidine-pyrimidine(Krieg et al, 1995 Nature 374:546-549; Pisetsky, 1996 J. Immunol.156:421-423; Hacker et al., 1998 EMBO J. 17:6230-6240; Lipford et al,1998 Trends in Microbiol. 6:496-500). However, it is now clear thatmouse lymphocytes respond quite well to phosphodiester CpG motifs thatdo not follow this “formula” (Yi et al., 1998 J. Immunol. 160:5898-5906)and the same is true of human B cells and dendritic cells (Hartmann etal, 1999 Proc. Natl. Acad. Sci USA 96:9305-10; Liang, 1996 J. Clin.Invest. 98:1119-1129).

[0006] Several past investigators have looked at whether the nucleotidecontent of ODN may have effects independently of the sequence of theODN. Interestingly, antisense ODN have been found to be generallyenriched in the content of GG, CCC, CC, CAC, and CG sequences, whilehaving reduced frequency of TT or TCC nucleotide sequences compared towhat would be expected if base usage were random (Smetsers et al., 1996Antisense Nucleic Acid Drug Develop. 6:63-67). This raised thepossibility that the over-represented sequences may comprise preferredtargeting elements for antisense oligonucleotides or visa versa. Onereason to avoid the use of thymidine-rich ODN for antisense experimentsis that degradation of the ODN by nucleases present in cells releasesfree thymidine which competes with ³H-thymidine which is frequently usedin experiments to assess cell proliferation (Matson et al., 1992Antisense Research and Development 2:325-330).

SUMMARY OF THE INVENTION

[0007] The invention is based in part on the surprising discovery that anew family of nucleic acids that induce higher levels of immunestimulation than previously known nucleic acids. This finding wassurprising in part because more than 100 nucleic acid sequences werescreened prior to discovering those disclosed herein.

[0008] The invention provides in one aspect, a composition comprising animmunostimulatory nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:1.

[0009] The invention further provides in another aspect, a method forstimulating an immune response in a subject in need thereof comprisingadministering to a subject an immunostimulatory nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, in an amounteffective to stimulate an immune response.

[0010] Various embodiments of the invention apply equally to the aspectsprovided herein and some of these are recited below.

[0011] In one embodiment, the immunostimulatory nucleic acid moleculeconsists of the nucleotide sequence of SEQ ID NO:1.

[0012] In another embodiment, the composition further comprises anantigen. Alternatively, the subject to be treated is furtheradministered an antigen. The antigen may be selected from the groupconsisting of a microbial antigen, a self antigen, a cancer antigen, andan allergen, but it is not so limited. In one embodiment, the microbialantigen is selected from the group consisting of a bacterial antigen, aviral antigen, a fungal antigen and a parasitic antigen. In anotherembodiment, the antigen is encoded by a nucleic acid vector. In arelated embodiment, the nucleic acid vector is separate from theimmunostimulatory nucleic acid. The antigen may be a peptide antigen.

[0013] In another embodiment, the composition further comprises anadjuvant, or the subject is further administered an adjuvant. Theadjuvant may be a mucosal adjuvant, but it is not so limited.

[0014] In another embodiment, the composition further comprises acytokine, or the subject is further administered a cytokine.

[0015] In still another embodiment, the composition further comprises atherapeutic agent selected from the group consisting of ananti-microbial agent, an anti-cancer agent, and an allergy/asthmamedicament, or the subject is further administered a therapeutic agentselected from the same group. In a related embodiment, theanti-microbial agent is selected from the group consisting of ananti-bacterial agent, an anti-viral agent, an anti-fungal agent, and ananti-parasite agent. In another related embodiment, the anti-canceragent is selected from the group consisting of a chemotherapeutic agent,a cancer vaccine, and an immunotherapeutic agent. In still anotherrelated embodiment, the allergy/asthma medicament is selected from thegroup consisting of PDE-4 inhibitor, bronchodilator/beta-2 agonist, K+channel opener, VLA-4 antagonist, neurokin antagonist, TXA2 synthesisinhibitor, xanthanine, arachidonic acid antagonist, 5 lipoxygenaseinhibitor, thromboxin A2 receptor antagonist, thromboxane A2 antagonist,inhibitor of 5-lipox activation protein, and protease inhibitor.

[0016] The immunostimulatory nucleic acid may in some embodiments have anucleotide backbone which includes at least one backbone modification.In one embodiment, the backbone modification is a phosphorothioatemodification. In another embodiment, the nucleotide backbone ischimeric. In one embodiment, the nucleotide backbone is entirelymodified.

[0017] In one embodiment, the composition further comprises apharmaceutically acceptable carrier.

[0018] In one embodiment, the immunostimulatory nucleic acid is free ofmethylated CpG dinucleotides. In another embodiment, theimmunostimulatory nucleic acid includes at least four CpG motifs. In yetanother embodiment, the immunostimulatory nucleic acid is T-rich. In arelated embodiment, the immunostimulatory nucleic acid includes a poly-Tsequence. In another embodiment, the immunostimulatory nucleic acidincludes a poly-G sequence.

[0019] In certain embodiments, the immunostimulatory nucleic acid isformulated in a variety of ways. In one embodiment, theimmunostimulatory nucleic acid is formulated for oral administration.The immunostimulatory nucleic acid may also be formulated as anutritional supplement. In a related embodiment, the nutritionalsupplement is formulated as a capsule, a pill, or a sublingual tablet.In another embodiment, the immunostimulatory nucleic acid is formulatedfor local administration. The immunostimulatory nucleic acid may also beformulated for parenteral administration or it may be formulated in asustained release device. The sustained release device may be amicroparticle but it is not so limited. In another embodiment, theimmunostimulatory nucleic acid is formulated for delivery to a mucosalsurface. The mucosal surface may be selected from the group consistingof an oral, nasal, rectal, vaginal, and ocular surface, but is not solimited.

[0020] In one embodiment, the immunostimulatory nucleic acid stimulatesa mucosal immune response. In another embodiment, the immunostimulatorynucleic acid stimulates a systemic immune response. In importantembodiments, the immunostimulatory nucleic acid stimulates both amucosal and systemic immune response. The immune response is anantigen-specific immune response, in some embodiments. In relatedembodiments, the immunostimulatory nucleic acid is provided in an amounteffective to stimulate a mucosal immune response. In other embodiments,the immunostimulatory nucleic acid is provided in an amount effective tostimulate a systemic immune response. In still other embodiments, theimmunostimulatory nucleic acid is provided in an amount effective tostimulate an innate immune response.

[0021] In various embodiments, the immunostimulatory nucleic acid isintended for treatment or prevention of a variety of diseases. Thus, inone embodiment, the immunostimulatory nucleic acid is provided in anamount effective to treat or prevent an infectious disease. In anotherembodiment, the immunostimulatory nucleic acid is provided in an amounteffective to treat or prevent an allergy. In still another embodiment,the immunostimulatory nucleic acid is provided in an amount effective totreat or prevent asthma. In yet a further embodiment, theimmunostimulatory nucleic acid is provided in an amount effective totreat or prevent a cancer.

[0022] In a related embodiment, the infectious disease is a herpessimplex virus infection. In another embodiment, the immunostimulatorynucleic acid is intended for administration to a subject that has or isat risk of developing an infection. The infection may be selected fromthe group consisting of a bacterial infection, a viral infection, afungal infection, and a parasite infection. In one embodiment, the viralinfection is selected from the group consisting of Humanimmunodeficiency viruses (HIV-1 and HIV-2), Human T lymphotrophic virustype I (HTLV-I), Human T lymphotrophic virus type II (HTLV-II), Herpessimplex virus type I (HSV-1), Herpes simplex virus type 2 (HSV-2), Humanpapilloma virus (multiple types), Hepatitis A virus, Hepatitis B virus,Hepatitis C and D viruses, Epstein-Barr virus (EBV), Cytomegalovirus andMolluscum contagiosum virus. In an important embodiment, the viralinfection is a herpes simplex virus infection.

[0023] In other embodiments, the infection is an infection with amicrobial species selected from the group consisting of herpesviridae,retroviridae, orthomyroviridae, toxoplasma, haemophilus, campylobacter,clostridium, E.coli, and staphylococcus. In related embodiments, theantigen to be administered to the subject or to be included in thecomposition is from one of the foregoing species.

[0024] In other embodiments, the immunostimulatory nucleic acid isintended from administration to a subject that has or is at risk ofdeveloping allergy, or a subject that has or is at risk of developingasthma, or a subject that has or is at risk of developing a cancer.

[0025] In embodiments relating to the treatment of a subject, the methodmay further comprise isolating an immune cell from the subject,contacting the immune cell with an effective amount to activate theimmune cell of the immunostimulatory nucleic acid and re-administeringthe activated immune cell to the subject. In one embodiment, the immunecell is a leukocyte. In another embodiment, the immune cell is adendritic cell. In another embodiment, the method further comprisescontacting the immune cell with an antigen.

[0026] In important embodiments, the subject is a human. In otherembodiments, the subject is selected from the group consisting of a dog,cat, horse, cow, pig, sheep, goat, chicken, monkey and fish.

[0027] Accordingly, the methods provided herein can be used on a subjectthat has or is at risk of developing an infectious disease and thereforethe method is a method for treating or preventing the infectiousdisease. The methods can also be used on a subject that has or is atrisk of developing asthma and the method is a method of treating orpreventing asthma in the subject. The method can also be used on asubject that has or is at risk of developing allergy and the method is amethod of treating or preventing allergy. And it can further be used ona subject that has or is at risk of developing a cancer and the methodis a method of treating or preventing the cancer. In one embodiment, thecancer is selected from the group consisting of biliary tract cancer;bone cancer; brain and CNS cancer; breast cancer; cervical cancer;choriocarcinoma; colon cancer; connective tissue cancer; endometrialcancer; esophageal cancer; eye cancer; gastric cancer; Hodgkin'slymphoma; intraepithelial neoplasms; larynx cancer; lymphomas; livercancer; lung cancer (e.g. small cell and non-small cell); melanoma;neuroblastomas; oral cavity cancer; ovarian cancer; pancreas cancer;prostate cancer; rectal cancer; sarcomas; skin cancer; testicularcancer; thyroid cancer; and renal cancer.

[0028] In yet another embodiment of the therapeutic or prophylacticmethods provided herein, the method may further comprise administeringan antibody specific for a cell surface antigen, and wherein the immuneresponse results in antigen dependent cellular cytotoxicity (ADCC).

[0029] The invention provides in another aspect, a method for preventingdisease in a subject, comprising administering to the subject animmunostimulatory nucleic acid on a regular basis to prevent disease inthe subject, wherein the immunostimulatory nucleic acid has a nucleotidesequence comprising SEQ ID NO:1.

[0030] In yet another aspect, the invention provides a method forinducing an innate immune response, comprising administering to thesubject an immunostimulatory nucleic acid in an amount effective foractivating an innate immune response, wherein the immunostimulatorynucleic acid has a nucleotide sequence comprising SEQ ID NO:1.

[0031] In still another aspect, the invention provides a method foridentifying an immunostimulatory nucleic acid comprising measuring acontrol level of activation of an immune cell population contacted withan immunostimulatory nucleic acid comprising a nucleotide sequence ofSEQ ID NO:1, measuring a test level of activation of an immune cellpopulation contacted with a test nucleic acid, and comparing the controllevel of activation to the test level of activation, wherein a testlevel that is equal to or above the control level is indicative of animmunostimulatory nucleic acid.

[0032] These and other aspects and embodiments of the invention will bedescribed in greater detail herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1: TLR9 engagement by ODNs 7909 and 10105. A TLR9-expressingcell line was incubated with the indicated concentrations of ODNs asdescribed in Materials and Methods. Shown is the mean Stimulation Indexabove media control for 4 independent experiments. IL-1 was used as apositive control for the reporter gene.

[0034]FIG. 2: B cells up regulate the activation marker CD86 uponincubation of PBMC with CpG ODNs. Human PBMC were incubated with ODNs7909 and 10105 as well as a control ODN at the indicated concentrationsfor 48 h. Shown is the mean percentage of CD86 expressing CD19-positiveB cells (measured by flow cytometry) of three different donors.

[0035]FIG. 3: Proliferation of B cells induced by CpG ODNs 7909 and10105. PBMC pre-incubated with the dye CFSE were cultured for 5 dayswithout or with the indicated ODN concentrations. Cells were harvestedand the decrease of the CFSE stain on proliferating CD19-positive Bcells was measured by flow cytometry (see also Materials and Methods).

[0036]FIG. 4: IFN-a secretion induced by ODNs 7909 and 10105. Human PBMCof three different donors were incubated with the indicatedconcentrations of ODNs for 48 h. The supernatant was harvested and IFN-αwas measured by ELISA (see Materials and Methods). Shown are the meanamounts of IFN-α obtained for the three different donors at eachconcentration.

[0037]FIG. 5: IP-10 secretion induced by ODNs 7909 and 10105. Human PBMCof three different donors were incubated with the indicatedconcentrations of ODNs for 48 h. The supernatant was harvested and IP-10was measured by ELISA (see Materials and Methods). Shown are the meanamounts of IP-10 obtained for the three different donors at eachconcentration.

[0038]FIG. 6: Time kinetic for IFN-α secretion. PBMC of two differentblood donors were incubated with the indicated concentrations of ODNs7909, 10105 or a control for 8 h or 24 h. Supernatants were harvestedand IFN-α measured by ELISA. Shown are the individual IFN-α amountsobtained at the different time points for the two donors.

[0039]FIG. 7: Time kinetic for IFN-α secretion. PBMC of two differentblood donors were incubated with the indicated concentrations of ODNs7909, 10105 or a control for 36 h or 48 h. Supernatants were harvestedand IFN-α measured by ELISA. Shown are the individual IFN-α amountsobtained at the different time points for the two donors.

[0040]FIG. 8: Time kinetic for IL-10 secretion. PBMC of three differentblood donors were incubated with the indicated concentrations of ODNs7909, 10105 or a control for 8 h, 24 h or 48 h. Supernatants wereharvested and IL-10 measured by ELISA. Shown are the individual IL-10amounts obtained at the different time points for the three donors.

[0041]FIG. 9: Time kinetic for IL-10 secretion. Shown is the sameexperiment as in FIG. 8. The mean amounts of IL-10 at each concentrationand time point between the three donors were calculated.

[0042]FIG. 10: Naïve BALB/c mouse splenocytes (5×10⁶/ml or 2.5×10⁶/ml)were incubated with media (negative control) or different amounts of CpGODN 7909 and 10105. Cells were pulsed with ³H-thymidine (20 μCi/ml) at96 hr post incubation for 16 hours, harvested and measured forradioactivity. Each bar represents the stimulation index (counts/min(CPM) of cells incubated/CPM of cells incubated with media).

[0043]FIG. 11: Naïve BALB/c mouse splenocytes (5×10⁶/ml) were incubatedwith media (negative control) or different amounts of CpG ODN 7909,10105 or control ODN 2137. Supernatants were harvested at 6 hr (forTNF-alpha, panel D), 24 hr (IL-12, panel B) or 48 hr (for IL-6, panel C,and IL-10, panel A).

[0044]FIG. 12: Naïve BALB/c mouse splenocytes (30×10⁶/ml) were incubatedwith media (negative control) or different amounts of CpG ODN 7909 and10105. NK activity was measured by using ⁵¹ Cr release assay.

[0045]FIG. 13: Adult (6-8 wk) BALB/c mice were immunized with 1 μg ofHBsAg alone or in combination with CpG ODN (10 μg) 10105, 7909 orcontrol ODN (10 μg) 2137. Animals were bled at 4 weeks post immunizationand plasma was assayed for total IgG levels against HBsAg (Anti-HBs).Each bar represents the geometric mean (±SEM) of the ELISA end pointdilution titer for the entire group (n=10). Titers were defined as thehighest dilution resulting in an absorbance value two times that ofnon-immune plasma with a cut-off value of 0.05.

[0046]FIG. 14: Adult BALB/c mice (6-8 wks old) were immunized with 1 μgof HBsAg alone or in combination with 10 μg CpG ODN 7909, 10105 or 10 μgcontrol ODN 2137. Animals were bled at 4 weeks post immunization andplasma was assayed for IgG1 and IgG2a levels against HBsAg (Anti-HBs).Each bar represents the geometric mean (±SEM) of the ELISA end pointdilution titer for the entire group (n=10). Titers were defined as thehighest dilution resulting in an absorbance value two times that ofnon-immune plasma with a cut-off value of 0.05.

DETAILED DESCRIPTION OF THE INVENTION

[0047] It was known in the prior art that CpG containing nucleic acidsstimulate the immune system can thereby be used to treat cancer,infectious diseases, allergy, asthma and other disorders, and to helpprotect against opportunistic infections following cancerchemotherapies. The strong yet balanced, cellular and humoral immuneresponses that result from CpG stimulation reflect the body's ownnatural defense system against invading pathogens and cancerous cells.CpG sequences, while relatively rare in human DNA, are commonly found inthe DNA of infectious organisms such as bacteria. The human immunesystem has apparently evolved to recognize CpG sequences as an earlywarning sign of infection, and to initiate an immediate and powerfulimmune response against invading pathogens without causing adversereactions frequently seen with other immune stimulatory agents. Thus,CpG containing nucleic acids, relying on this innate immune defensemechanism, can utilize a unique and natural pathway for immune therapy.

[0048] The effects of CpG nucleic acids on immune modulation werediscovered by the inventor of the instant patent application and havebeen described extensively in co-pending patent applications, such asU.S. patent application Ser. Nos: 08/386,063 filed on Feb. 2, 1995 (andrelated PCT US95/01570); 08/738,652 filed on Oct. 30, 1996; 08/960,774filed on Oct. 30, 1997 (and related PCT/US97/19791, WO 98/18810);09/191,170 filed on Nov. 13, 1998; 09/030,701 filed on Feb. 25, 1998(and related PCT/US98/03678; 09/082,649 filed on May 20, 1998 (andrelated PCT/US98/10408); 09/325,193 filed on Jun. 3, 1999 (and relatedPCT/US98/04703); 09/286,098 filed on Apr. 2, 1999 (and relatedPCT/US99/07335); 09/306,281 filed on May 6, 1999 (and relatedPCT/US99/09863). The entire contents of each of these patents and patentapplications is hereby incorporated by reference.

[0049] The invention is based, in part, on the unexpected discovery of afamily of nucleic acids that is as immunostimulatory as previouslyreported CpG nucleic acids. This family of nucleic acids comprises thenucleotide sequence having the formula of 5′ X₁X₂X₃ X₄X₅X₆ X₇X₈X₉ X₁₀X₁₁X₁₂ X₁₃X₁₄X₁₅ TTT TTT CGA 3′ (SEQ ID NO:3) wherein X₁, X₂, X₃, X₄, X₅,X₆, X_(7,) X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, and X₁₅ are independentlyselected residues that may be selected from the group of nucleotidesconsisting of adenosine, guanosine, thymidine, and cytosine. In someembodiments, there may be no flanking residues. Such a nucleic acidwould comprise a nucleotide sequence of 5′ TTT TTT CGA 3′ (SEQ ID NO:4).

[0050] In other embodiments, the nucleic acid may lack X₁; X₁ and X₂;X₁, X₂ and X₃; X₁, X₂, X₃ and X₄; or X₁, X₂, X₃, X₄ and X₅, X₁ throughX₆, X₁ through X₇, X₁ through X₈, X₁ through X₉, X₁ through X₁₀, X₁through X₁₁, X₁ through X₁₂, X₁ through X₁₃, X₁ through X₁₄, and X₁through X₁₅.

[0051] In various embodiments, X, is a thymidine, and/or X₂ is cytosine,and/or X₃ is a guanosine, and/or X₄ is a thymidine, and/or X₅ is acytosine, and/or X₆ is a guanosine, and/or X₇ is a thymidine, and/or X₈is a thymidine, and/or X₉ is a thymidine, and/or X₁₀ is a thymidine,and/or X₁₁ is a guanosine, and/or X₁₂ is a thymidine, and/or X₁₃ is acytosine, and/or X₁₄ is a guanosine, and/or X₁₅ is a thymidine. Those ofordinary skill in the art will be able to determine the sequence of theremaining nucleic acids belonging to this family.

[0052] The nucleic acids of this family are generally at least 9nucleotides in length. In some embodiments, the nucleic acids are atleast 10, at least 12, at least 15, at least 18, at least 20, at least22, and at least 24 nucleotides in length. In a preferred embodiment,the nucleic acids are 24 nucleotides in length. In still furtherembodiments, the nucleic acids are more than 24 nucleotides in length.Examples include nucleic acids that are at least 50, at least 75, atleast 100, at least 200, at least 500, at least 1000 nucleotides inlength, or longer. Preferably, the nucleic acids are 9-100, and morepreferably 24-100 nucleotides in length.

[0053] All the nucleic acids of this first family contain at least oneCpG motif. These nucleic acids may contain two, three, four or more CpGmotifs. The CpG motifs may be contiguous to each other, oralternatively, they may be spaced apart from each other at constant orrandom distances.

[0054] The nucleic acids of this family also contain anoverrepresentation of thymidine nucleotides. These nucleic acids maycontain at least 60%, at least 55%, or at least 50% thymidines.

[0055] The invention is further premised, in part, on the unexpecteddiscovery of another family of nucleic acids that is asimmunostimulatory as previously reported CpG nucleic acids. This familyof nucleic acids comprises the nucleotide sequence having the formula of5′ TCG TCG TTT TGT CGT TTT T X₁X₂ X₃X₄X₅ 3′ (SEQ ID NO:5) wherein X₁through X₉ are independently selected residues that may be selected fromthe group of nucleotides consisting of adenosine, guanosine, thymidine,and cytosine. In some embodiments, there may be no flanking residues. Asan example, the nucleic acid may comprise a nucleotide sequence of 5′TCG TCG TTT TGT CGT TTT T 3′ (SEQ ID NO:6).

[0056] In other embodiments, the nucleic acid may lack X₅; X₅ and X₄;X₅, X₄ and X₃; X₅ through X₂; and X₅ through X₁.

[0057] In various embodiments, X₁ is a thymidine, and/or X₂ isthymidine, and/or X₃ is a cytosine, and/or X₄ is a guanosine, and/or X₅is an adenine. Those of ordinary skill in the art will be able todetermine the sequence of the remaining nucleic acids belonging to thisfamily.

[0058] The nucleic acids of this family are generally at least 19nucleotides in length. In some embodiments, the nucleic acids are atleast 20, at least 22, and at least 24 nucleotides in length. In apreferred embodiment, the nucleic acids are 24 nucleotides in length. Instill further embodiments, the nucleic acids are more than 24nucleotides in length. Examples include nucleic acids that are at least50, at least 75, at least 100, at least 200, at least 500, at least 1000nucleotides in length, or longer. Preferably, the nucleic acids are19-100, and more preferably 24-100 nucleotides in length.

[0059] All the nucleic acids of this second family contain at leastthree CpG motifs. These nucleic acids may contain four or more CpGmotifs, depending upon the embodiment. The CpG motifs may be contiguousto each other, or alternatively, they may be spaced apart from eachother at constant or random distances.

[0060] The nucleic acids of this family also contain anoverrepresentation of thymidine nucleotides. These nucleic acids maycontain at least 60%, at least 55%, or at least 50% thymidines.

[0061] In another aspect, the invention provides a nucleic acidcomprising the nucleotide sequence of TCG TCG TTT TGT CGT TTT TTT CGA(SEQ ID NO:1). As described in greater detail in the Examples, thisnucleic acid was identified only after screening a multitude of nucleicacids for those having similar or greater immunostimulatory activitythan previously identified immunostimulatory nucleic acids. Morespecifically, the nucleic acids were compared to a nucleic acid having anucleotide sequence of TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO:2)that was previously shown to be immunostimulatory. The nucleic acidcomprising SEQ ID NO:1 was identified only after screening approximately165 nucleic acids for those having immunostimulatory capacity similar toor greater than that of nucleic acids comprising SEQ ID NO:2. Thedifference in activity is surprising because there is 79% identitybetween SEQ ID NO:1 and SEQ ID NO:2 (i.e., five of the last 3′nucleotides differ between SEQ ID NO:1 and SEQ ID NO:2). It wasunexpected that such a change in sequence would result in an increase inimmunostimulation.

[0062] In yet other aspects of the invention, nucleic acids having thefollowing nucleotide sequences are provided: 5′ TCG TCG TTT TGT CGT TTTTTT CG 3′ (SEQ ID NO:7); 5′ TCG TCG TTT TGT CGT TTT TTT C 3′; (SEQ IDNO:8) 5′ TCG TCG TTT TGT CGT TTT TTT 3′; (SEQ ID NO:9) 5′ TCG TCG TTTTGT CGT TTT TT 3′; (SEQ ID NO:10) 5′ CG TCG TTT TGT CGT TTT TTT (SEQ IDNO:11) CGA 3′; 5′ G TCG TTT TGT CGT TTT TTT CGA 3′; (SEQ ID NO:12) 5′TCG TTT TGT CGT TTT TTT CGA 3′; (SEQ ID NO:13) 5′ CG TTT TGT CGT TTT TTTCGA 3′; (SEQ ID NO:14) 5′ G TTT TGT CGT TTT TTT CGA 3′; (SEQ ID NO:15)5′ TTT TGT CGT TTT TTT CGA 3′; (SEQ ID NO:16) 5′ TT TGT CGT TTT TTT CGA3′; (SEQ ID NO:17) 5′ T TGT CGT TTT TTT CGA 3′; (SEQ ID NO:18) 5′ TGTCGT TTT TTT CGA 3′; (SEQ ID NO:19) 5′ GT CGT TTT TTT CGA 3′; (SEQ IDNO:20) 5′ T CGT TTT TTT CGA 3′; (SEQ ID NO:21) 5′ CGT TTT TTT CGA 3′;(SEQ ID NO:22) 5′ GT TTT TTT CGA 3′; (SEQ ID NO:23) and 5′ T TTT TTT CGA3′. (SEQ ID NO:24)

[0063] These immunostimulatory nucleic acids are capable of activatingthe innate immune system, and augmenting both humoral and cellularantigen specific responses when co-administered with an antigen, such asHepatitis B surface antigen. The Examples provided herein demonstratethat these nucleic acids can stimulate human immune cells in vitro, andmurine cells in vitro and in vivo. When compared to a sequence known tobe a potent adjuvant, the nucleic acid of SEQ ID NO:1 is found to workas well or better as a vaccine adjuvant.

[0064] The CpG motifs of the nucleic acids described herein arepreferably unmethylated. An unmethylated CpG motif is an unmethylatedcytosine-guanine dinucleotide sequence (i.e. an unmethylated 5′ cytosinefollowed by 3′ guanosine and linked by a phosphate bond). All thenucleic acid described herein are immunostimulatory. In some embodimentsof the invention, the CpG motifs are methylated. A methylated CpG motifis a methylated cytosine-guanine dinucleotide sequence (i.e., amethylated 5′ cytosine followed by a 3′ guanosine and linked by aphosphate bond).

[0065] A CpG nucleic acid is a nucleic acid that comprises the formula

5′ X₁ X₂CGX₃ X₄ 3′

[0066] wherein C is unmethylated, wherein X₁X₂ and X₃X₄ are nucleotides.In a related embodiment, the 5′ X₁ X₂CGX₃ X₄ 3′ sequence is anon-palindromic sequence. In certain embodiments, X₁X₂ are nucleotidesselected from the group consisting of GpT, GpG, GpA, ApA, ApT, ApG, CpT,CpA, CpG, TpA, TpT, and TpG; and X₃X₄ are nucleotides selected from thegroup consisting of TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA,ApA, and CpA. In more particular embodiments, X₁X₂ are nucleotidesselected from the group consisting of GpA and GpT; and X₃X₄ are TpT. Inyet other embodiments, X₁X₂ are both purines and X₃X₄ are bothpyrimidines. In another embodiment, X₂ is a T and X₃ is a pyrimidine.Examples of CpG nucleic acids are described in U.S. Non-ProvisionalPatent Application Serial No. 09/669,187, filed Sep. 25, 2000, and inpublished PCT Patent Application PCT/US00/26383, having publicationnumber WO01/22972.

[0067] The nucleic acids of the invention can further contain otherimmunostimulatory motifs such as poly T motifs, poly G motifs, TGmotifs, poly A motifs, poly C motifs, and the like, provided that thecore sequences of SEQ ID NO:4 and SEQ ID NO:6 are present. Theseimmunostimulatory motifs are described in greater detail below or inU.S. Non-Provisional Patent Application Serial No. 09/669,187, filedSep. 25, 2000, and published PCT Patent Application PCT/US00/26383,having publication number WO01/22972

[0068] A T-rich nucleic acid is a nucleic acid which includes at leastone poly T sequence and/or which has a nucleotide composition of greaterthan 25 % T nucleotide residues. A nucleic acid having a poly-T sequenceincludes at least four Ts in a row, such as 5′TTTT3′. Preferably aT-rich nucleic acid includes more than one poly T sequence. In preferredembodiments the T-rich nucleic acid may have 2, 3, 4, etc poly Tsequences. Other T-rich nucleic acids according to the invention have anucleotide composition of greater than 25% T nucleotide residues, but donot necessarily include a poly T sequence. In these T-rich nucleic acidsthe T nucleotide resides may be separated from one another by othertypes of nucleotide residues, i.e., G, C, and A. In some embodiments theT-rich nucleic acids have a nucleotide composition of greater than 35%,40%, 50%, 60%, 70%, 80%, 90%, and 99%, T nucleotide residues and everyinteger % in between. Preferably the T-rich nucleic acids have at leastone poly T sequence and a nucleotide composition of greater than 25% Tnucleotide residues.

[0069] Poly G nucleic acids preferably are nucleic acids having thefollowing formulas:

5′ X₁X₂GGGX₃X₄3′

[0070] wherein X₁, X₂, X₃, and X₄ are nucleotides. In preferredembodiments at least one of X₃ and X₄ are a G. In other embodiments bothof X₃ and X₄ are a G. In yet other embodiments the preferred formula is5′ GGGNGGG3′, or 5′ GGGNGGGNGGG3′ wherein N represents between 0 and 20nucleotides.

[0071] A C-rich nucleic acid is a nucleic acid molecule having at leastone or preferably at least two poly-C regions or which is composed of atleast 50% C nucleotides. A poly-C region is at least four C residues ina row. Thus a poly-C region is encompassed by the formula 5′CCCC 3′. Insome embodiments it is preferred that the poly-C region have the formula5′CCCCCC3′. Other C-rich nucleic acids according to the invention have anucleotide composition of greater than 50% C nucleotide residues, but donot necessarily include a poly C sequence. In these C-rich nucleic acidsthe C nucleotide residues may be separated from one another by othertypes of nucleotide residues, i.e., G, T, and A. In some embodiments theC-rich nucleic acids have a nucleotide composition of greater than 60%,70%, 80%, 90%, and 99%, C nucleotide residues and every integer % inbetween. Preferably the C-rich nucleic acids have at least one poly Csequence and a nucleotide composition of greater than 50% C nucleotideresidues, and in some embodiments are also T-rich.

[0072] The immunostimulatory nucleic acids can be double-stranded orsingle-stranded. Generally, double-stranded molecules are more stable invivo, while single-stranded molecules have increased immune activity.Thus in some aspects of the invention it is preferred that the nucleicacid be single stranded and in other aspects it is preferred that thenucleic acid be double stranded.

[0073] The terms “nucleic acid” and “oligonucleotide” are usedinterchangeably herein to mean multiple nucleotides (i.e. moleculescomprising a sugar (e.g. ribose or deoxyribose) linked to a phosphategroup and to an exchangeable organic base, which is either a substitutedpyrimidine (e.g. cytosine (C), thymidine (T) or uracil (U)) or asubstituted purine (e.g. adenine (A) or guanine (G)). As used herein,the terms refer to oligoribonucleotides as well asoligodeoxyribonucleotides. The terms shall also include polynucleosides(i.e. a polynucleotide minus the phosphate) and any other organic basecontaining polymer. Nucleic acid molecules can be obtained from existingnucleic acid sources (e.g., genomic or cDNA), but are preferablysynthetic (e.g. produced by nucleic acid synthesis).

[0074] The immunostimulatory oligonucleotides of the instant inventioncan encompass various chemical modifications and substitutions, incomparison to natural RNA and DNA, involving a phosphodiesterinternucleoside bridge, a β-D-ribose unit and/or a natural nucleosidebase (adenine, guanine, cytosine, thymine, uracil). Examples of chemicalmodifications are known to the skilled person and are described, forexample, in Uhlmann E et al. (1990) Chem Rev 90:543; “Protocols forOligonucleotides and Analogs” Synthesis and Properties & Synthesis andAnalytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993;Crooke ST et al. (1996) Annu Rev Pharmacol Toxicol 36:107-129; andHunziker J et al. (1995) Mod Synth Methods 7:331-417. An oligonucleotideaccording to the invention may have one or more modifications, whereineach modification is located at a particular phosphodiesterinternucleoside bridge and/or at a particular β-D-ribose unit and/or ata particular natural nucleoside base position in comparison to anoligonucleotide of the same sequence which is composed of natural DNA orRNA.

[0075] For example, the oligonucleotides may comprise one or moremodifications and wherein each modification is independently selectedfrom:

[0076] a) the replacement of a phosphodiester internucleoside bridgelocated at the 3′ and/or the 5′ end of a nucleoside by a modifiedinternucleoside bridge,

[0077] b) the replacement of phosphodiester bridge located at the 3′and/or the 5′ end of a nucleoside by a dephospho bridge,

[0078] c) the replacement of a sugar phosphate unit from the sugarphosphate backbone by another unit,

[0079] d) the replacement of a β-D-ribose unit by a modified sugar unit,and

[0080] e) the replacement of a natural nucleoside base by a modifiednucleoside base.

[0081] More detailed examples for the chemical modification of anoligonucleotide are as follows.

[0082] Nucleic acids also include substituted purines and pyrimidinessuch as C-5 propyne pyrimidine and 7-deaza-7-substituted purine modifiedbases. Wagner RW et al. (1996) Nat Biotechnol 14:840-4. Purines andpyrimidines include but are not limited to adenine, cytosine, guanine,thymidine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine,2,6-diaminopurine, hypoxanthine, and other naturally and non-naturallyoccurring nucleobases, substituted and unsubstituted aromatic moieties.Other such modifications are well known to those of skill in the art. Inall of the foregoing embodiments, an X residue can also be anon-naturally occurring nucleotide, or a nucleotide analog, such asthose described herein.

[0083] A modified base is any base which is chemically distinct from thenaturally occurring bases typically found in DNA and RNA such as T, C,G, A, and U, but which share basic chemical structures with thesenaturally occurring bases. The modified nucleoside base may be, forexample, selected from hypoxanthine, uracil, dihydrouracil,pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil,5-(C₁-C₆)-alkyluracil, 5-(C₂-C₆)-alkenyluracil, 5-(C₂-C₆)-alkynyluracil,5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil,5-hydroxycytosine, 5-(C₁-C₆)-alkylcytosine, 5-(C₂-C₆)-alkenylcytosine,5-(C₂-C₆)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine,5-bromocytosine, N²-dimethylguanine, 2,4-diamino-purine, 8-azapurine, asubstituted 7-deazapurine, preferably 7-deaza-7-substituted and/or7-deaza-8-substituted purine, 5-hydroxymethylcytosine, N4-alkylcytosine,e.g., N4-ethylcytosine, 5-hydroxydeoxycytidine,5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine, e.g.,N4-ethyldeoxycytidine, 6-thiodeoxyguanosine, and deoxyribonucleosides ofnitropyrrole, C5-propynylpyrimidine, and diaminopurine e.g.,2,6-diaminopurine, inosine, 5-methylcytosine, 2-aminopurine,2-amino-6-chloropurine, hypoxanthine or other modifications of a naturalnucleoside bases. This list is meant to be exemplary and is not to beinterpreted to be limiting.

[0084] In particular formulas described herein a set of modified basesis defined. For instance the letter Y is used to refer to a nucleotidecontaining a cytosine or a modified cytosine. A modified cytosine asused herein is a naturally occurring or non-naturally occurringpyrimidine base analog of cytosine which can replace this base withoutimpairing the immunostimulatory activity of the oligonucleotide.Modified cytosines include but are not limited to 5-substitutedcytosines (e.g. 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine,5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine,5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstitutedor substituted 5-alkynyl-cytosine), 6-substituted cytosines,N4-substituted cytosines (e.g. N4-ethyl-cytosine), 5-aza-cytosine,2-mercapto-cytosine, isocytosine, pseudo-isocytosine, cytosine analogswith condensed ring systems (e.g. N,N′-propylene cytosine orphenoxazine), and uracil and its derivatives (e.g. 5-fluoro-uracil,5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil,5-propynyl-uracil). Some of the preferred cytosines include5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine,5-hydroxymethyl-cytosine, and N4-ethyl-cytosine. In another embodimentof the invention, the cytosine base is substituted by a universal base(e.g. 3-nitropyrrole, P-base), an aromatic ring system (e.g.fluorobenzene or difluorobenzene) or a hydrogen atom (dSpacer). Theletter Z is used to refer to guanine or a modified guanine base. Amodified guanine as used herein is a naturally occurring ornon-naturally occurring purine base analog of guanine which can replacethis base without impairing the immunostimulatory activity of theoligonucleotide. Modified guanines include but are not limited to7-deazaguanine, 7-deaza-7-substituted guanine (such as7-deaza-7-(C2-C6)alkynylguanine), 7-deaza-8-substituted guanine,hypoxanthine, N2-substituted guanines (e.g. N2-methyl-guanine),5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione,2,6-diaminopurine, 2-aminopurine, purine, indole, adenine, substitutedadenines (e.g. N6-methyl-adenine, 8-oxo-adenine) 8-substituted guanine(e.g. 8-hydroxyguanine and 8-bromoguanine), and 6-thioguanine. Inanother embodiment of the invention, the guanine base is substituted bya universal base (e.g. 4-methyl-indole, 5-nitro-indole, and K-base), anaromatic ring system (e.g. benzimidazole or dichloro-benzimidazole,1-methyl-1H-[1,2,4]triazole-3-carboxylic acid amide) or a hydrogen atom(dSpacer).

[0085] The oligonucleotides may include modified internucleotidelinkages, such as those described in a or b above. These modifiedlinkages may be partially resistant to degradation (e.g., arestabilized). A “stabilized nucleic acid molecule” shall mean a nucleicacid molecule that is relatively resistant to in vivo degradation (e.g.via an exo- or endo-nuclease). Stabilization can be a function of lengthor secondary structure. Nucleic acids that are tens to hundreds ofkilobases long are relatively resistant to in vivo degradation. Forshorter nucleic acids, secondary structure can stabilize and increasetheir effect. For example, if the 3′ end of an nucleic acid hasself-complementarity to an upstream region, so that it can fold back andform a sort of stem loop structure, then the nucleic acid becomesstabilized and therefore exhibits more activity.

[0086] Nucleic acid stabilization can also be accomplished via phosphatebackbone modifications. Oligonucleotides having phosphorothioatelinkages, in some embodiments, may provide maximal activity and protectthe oligonucleotide from degradation by intracellular exo- andendo-nucleases.

[0087] It has been demonstrated that modification of the nucleic acidbackbone provides enhanced activity of nucleic acids when administeredin vivo. Constructs having phosphorothioate linkages provide maximalactivity and protect the nucleic acid from degradation by intracellularexo- and endo-nucleases. Other modified nucleic acids includephosphodiester modified nucleic acids, combinations of phosphodiesterand phosphorothioate nucleic acid, methylphosphonate,methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinationsthereof. Each of these combinations and their particular effects onimmune cells is discussed in more detail with respect to CpG nucleicacids in PCT Published Patent Applications PCT/US95/01570 (WO 96/02555)and PCT/US97/19791 (WO 98/18810) and in U.S. Pat. Nos. U.S. Pat. No.6,194,388 B1 issued Feb. 27, 2001 and U.S. Pat. No. 6,239,116 B1 issuedMay 29, 2001, the entire contents of which are hereby incorporated byreference. It is believed that these modified nucleic acids may showmore stimulatory activity due to enhanced nuclease resistance, increasedcellular uptake, increased protein binding, and/or altered intracellularlocalization.

[0088] Other stabilized nucleic acids include: nonionic DNA analogs,such as alkyl- and aryl-phosphates (in which the charged phosphonateoxygen is replaced by an alkyl or aryl group), phosphodiester andalkylphosphotriesters, in which the charged oxygen moiety is alkylated.Nucleic acids which contain diol, such as tetraethyleneglycol orhexaethyleneglycol, at either or both termini have also been shown to besubstantially resistant to nuclease degradation.

[0089] The oligonucleotides may have one or two accessible 5′ ends. Itis possible to create modified oligonucleotides having two such 5′ ends,for instance, by attaching two oligonucleotides through a 3′-3′ linkageto generate an oligonucleotide having one or two accessible 5′ ends. The3′-3′-linkage may be a phosphodiester, phosphorothioate or any othermodified internucleoside bridge. Methods for accomplishing such linkagesare known in the art. For instance, such linkages have been described inSeliger, H. et al., Oligonucleotide analogs with terminal 3′-3′- and5′-5′-internucleotidic linkages as antisense inhibitors of viral geneexpression, Nucleosides & Nucleotides (1991), 10(1-3), 469-77 and Jiang,et al., Pseudo-cyclic oligonucleotides: in vitro and in vivo properties,Bioorganic & Medicinal Chemistry (1999), 7(12), 2727-2735.

[0090] Additionally, 3′3′-linked ODNs where the linkage between the3′-terminal nucleosides is not a phosphodiester, phosphorothioate orother modified bridge, can be prepared using an additional spacer, suchas tri- or tetra-ethylenglycol phosphate moiety (Durand, M. et al,Triple-helix formation by an oligonucleotide containing one (dA)12 andtwo (dT)12 sequences bridged by two hexaethylene glycol chains,Biochemistry (1992), 31(38), 9197-204, U.S. Pat. No. 5,658,738, and U.S.Pat. No. 5,668,265). Alternatively, the non-nucleotidic linker may bederived from ethanediol, propanediol, or from an abasic deoxyribose(dSpacer) unit (Fontanel, Marie Laurence et al., Sterical recognition byT4 polynucleotide kinase of non-nucleosidic moieties 5′-attached tooligonucleotides; Nucleic Acids Research (1994), 22(11), 2022-7) usingstandard phosphoramidite chemistry. The non-nucleotidic linkers can beincorporated once or multiple times, or combined with each otherallowing for any desirable distance between the 3′-ends of the two ODNsto be linked.

[0091] A phosphodiester intemucleoside bridge located at the 3′ and/orthe 5′ end of a nucleoside can be replaced by a modified internucleosidebridge, wherein the modified internucleoside bridge is for exampleselected from phosphorothioate, phosphorodithioate,NR¹R²-phosphoramidate, boranophosphate, α-hydroxybenzyl phosphonate,phosphate-(C₁-C₂₁)—O-alkyl ester,phosphate-[(C₆-C₁₂)aryl-(C₁-C₁₂)—O-alkyl]ester, (C₁-C₈)alkylphosphonateand/or (C₆-C₁₂)arylphosphonate bridges, (C₇-C₁₂)-α-hydroxymethyl-aryl(e.g., disclosed in WO 95/01363), wherein (C₆-C₁₂)aryl, (C₆-C₂₀)aryl and(C₆-C₁₄)aryl are optionally substituted by halogen, alkyl, alkoxy,nitro, cyano, and where R¹ and R² are, independently of each other,hydrogen, (C₁-C₈)-alkyl, (C₆-C₂₀)-aryl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl,preferably hydrogen, (C₁-C₈)-alkyl, preferably (C₁-C₄)-alkyl and/ormethoxyethyl, or R¹ and R² form, together with the nitrogen atomcarrying them, a 5-6-membered heterocyclic ring which can additionallycontain a further heteroatom from the group O, S and N.

[0092] The replacement of a phosphodiester bridge located at the 3′and/or the 5′ end of a nucleoside by a dephospho bridge (dephosphobridges are described, for example, in Uhlmann E and Peyman A in“Methods in Molecular Biology”, Vol. 20, “Protocols for Oligonucleotidesand Analogs”, S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16,pp. 355 ff), wherein a dephospho bridge is for example selected from thedephospho bridges formacetal, 3′-thioformacetal, methylhydroxylamine,oxime, methylenedimethyl-hydrazo, dimethylenesulfone and/or silylgroups.

[0093] The compositions of the invention may optionally be have chimericbackbones. As used herein, a chimeric backbone is one that comprisesmore than one type of linkage. In one embodiment, the chimeric backbonecan be represented by the formula: 5′ Y₁N₁ZN₂Y₂ 3′. Y₁ and Y₂ arenucleic acid molecules having between 1 and 10 nucleotides. Y₁ and Y₂each include at least one modified internucleotide linkage. Since atleast 2 nucleotides of the chimeric oligonucleotides include backbonemodifications these nucleic acids are an example of one type of“stabilized immunostimulatory nucleic acids.”

[0094] With respect to the chimeric oligonucleotides, Y₁ and Y₂ areconsidered independent of one another. This means that each of Y₁ and Y₂may or may not have different sequences and different backbone linkagesfrom one anther in the same molecule. In some embodiments Y₁ and/or Y₂have between 3 and 8 nucleotides. N₁ and N₂ are nucleic acid moleculeshaving between 0 and 5 nucleotides as long as N₁ZN₂ has at least 6nucleotides in total. The nucleotides of N₁ZN₂ have a phosphodiesterbackbone and do not include nucleic acids having a modified backbone. Zis an immunostimulatory nucleic acid motif, preferably selected fromthose recited herein.

[0095] The center nucleotides (N₁ZN₂) of the formula Y₁N₁ZN₂Y₂ havephosphodiester internucleotide linkages and Y₁ and Y₂ have at least one,but may have more than one or even may have all modified internucleotidelinkages. In preferred embodiments Y₁ and/or Y₂ have at least two orbetween two and five modified internucleotide linkages or Y₁ has twomodified internucleotide linkages and Y₂ has five modifiedinternucleotide linkages or Y₁ has five modified internucleotidelinkages and Y₂ has two modified internucleotide linkages. The modifiedinternucleotide linkage, in some embodiments is a phosphorothioatemodified linkage, a phosphorodithioate modified linkage or a p-ethoxymodified linkage.

[0096] The nucleic acids also include nucleic acids having backbonesugars which are covalently attached to low molecular weight organicgroups other than a hydroxyl group at the 2′ position and other than aphosphate group at the 5′ position. Thus, modified nucleic acids mayinclude a 2′-O-alkylated ribose group. In addition, modified nucleicacids may include sugars such as arabinose or 2′-fluoroarabinose insteadof ribose. Thus the nucleic acids may be heterogeneous in backbonecomposition thereby containing any possible combination of polymer unitslinked together such as peptide-nucleic acids (which have amino acidbackbone with nucleic acid bases). In some embodiments, the nucleicacids are homogeneous in backbone composition. Other examples aredescribed in more detail below.

[0097] A sugar phosphate unit (i.e., a β-D-ribose and phosphodiesterinternucleoside bridge together forming a sugar phosphate unit) from thesugar phosphate backbone (i.e., a sugar phosphate backbone is composedof sugar phosphate units) can be replaced by another unit, wherein theother unit is for example suitable to build up a “morpholino-derivative”oligomer (as described, for example, in Stirchak EP et al. (1989)Nucleic Acids Res 17:612941), that is, e.g., the replacement by amorpholino-derivative unit; or to build up a polyamide nucleic acid(“PNA”; as described for example, in Nielsen PE et al. (1994) BioconjugChem 5:3-7), that is, e.g., the replacement by a PNA backbone unit,e.g., by 2-aminoethylglycine. The oligonucleotide may have othercarbohydrate backbone modifications and replacements, such as peptidenucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA),and oligonucleotides having backbone sections with alkyl linkers oramino linkers. The alkyl linker may be branched or unbranched,substituted or unsubstituted, and chirally pure or a racemic mixture.

[0098] A β-ribose unit or a β-D-2′-deoxyribose unit can be replaced by amodified sugar unit, wherein the modified sugar unit is for exampleselected from β-D-ribose, α-D-2′-deoxyribose, L-2′-deoxyribose,2′-F-2′-deoxyribose, 2′-F-arabinose, 2′-O-(C₁-C₆)alkyl-ribose,preferably 2′-O—(C₁-C₆)alkyl-ribose is 2′-O-methylribose,2′-O—(C₂-C₆)alkenyl-ribose, 2′-[O-(C₁-C₆)alkyl-O—(C₁-C₆)alkyl]-ribose,2′-NH₂-2′-deoxyribose, β-D-xylo-furanose, α-arabinofuranose,2,4-dideoxy-β-D-erythro-hexo-pyranose, and carbocyclic (described, forexample, in Froehler J (1992) Am Chem Soc 114:8320) and/or open-chainsugar analogs (described, for example, in Vandendriessche et al. (1993)Tetrahedron 49:7223) and/or bicyclosugar analogs (described, forexample, in Tarkov M et al. (1993) Helv Chim Acta 76:481).

[0099] In some embodiments the sugar is 2′-O-methylribose, particularlyfor one or both nucleotides linked by a phosphodiester orphosphodiester-like internucleoside linkage.

[0100] For use in the instant invention, the oligonucleotides of theinvention can be synthesized de novo using any of a number of procedureswell known in the art. For example, the b-cyanoethyl phosphoramiditemethod (Beaucage, S. L., and Caruthers, M. H., Tet. Let. 22:1859, 1981);nucleoside H-phosphonate method (Garegg et al., Tet. Let. 27:4051-4054,1986; Froehler et al., Nucl. Acid. Res. 14:5399-5407, 1986, ; Garegg etal., Tet. Let. 27:4055-4058, 1986, Gaffney et al., Tet. Let.29:2619-2622, 1988). These chemistries can be performed by a variety ofautomated nucleic acid synthesizers available in the market. Theseoligonucleotides are referred to as synthetic oligonucleotides.Alternatively, T-rich and/or TG dinucleotides can be produced on a largescale in plasmids, (see Sambrook, T., et al., “Molecular Cloning: ALaboratory Manual”, Cold Spring Harbor laboratory Press, New York, 1989)and separated into smaller pieces or administered whole. Nucleic acidscan be prepared from existing nucleic acid sequences (e.g., genomic orcDNA) using known techniques, such as those employing restrictionenzymes, exonucleases or endonucleases.

[0101] Modified backbones such as phosphorothioates may be synthesizedusing automated techniques employing either phosphoramidate orH-phosphonate chemistries. Aryl-and alkyl-phosphonates can be made,e.g., as described in U.S. Pat. No. 4,469,863; and alkylphosphotriesters(in which the charged oxygen moiety is alkylated as described in U.S.Pat. No. 5,023,243 and European Patent No. 092,574) can be prepared byautomated solid phase synthesis using commercially available reagents.Methods for making other DNA backbone modifications and substitutionshave been described (e.g., Uhlmann, E. and Peyman, A., Chem. Rev.90:544, 1990; Goodchild, J., Bioconjugate Chem. 1:165, 1990).

[0102] Nucleic acids prepared in this manner are referred to as isolatednucleic acid. An “isolated nucleic acid” generally refers to a nucleicacid which is separated from components with which it is normallyassociated in nature. As an example, an isolated nucleic acid may be onewhich is separated from a cell, from a nucleus, from mitochondria orfrom chromatin.

[0103] In the case where the nucleic acid is administered in conjunctionwith an antigen that is encoded in a nucleic acid vector (as describedherein), it is preferred that the backbone of the nucleic acid be achimeric combination of phosphodiester and phosphorothioate (or otherphosphate modification). The cell may have a problem taking up a plasmidvector in the presence of completely phosphorothioate nucleic acid. Thuswhen both a vector and a nucleic acid are delivered to a subject, it ispreferred that the nucleic acid have a chimeric backbone or have aphosphorothioate backbone but that the plasmid be associated with avehicle that delivers it directly into the cell, thus avoiding the needfor cellular uptake. Such vehicles are known in the art and include, forexample, liposomes and gene guns.

[0104] The invention further embraces the use of any of these foregoingnucleic acids in the methods recited herein, as well as all previouslydescribed and previously known uses of immunostimulatory nucleic acids.

[0105] It has been discovered according to the invention that theimmunostimulatory nucleic acids have surprisingly increased immunestimulatory effects. For example, it has been demonstrated that thenucleic acids described herein are able to provide protection againstinfection, probably by generally stimulating the immune system. TheExamples illustrate the ability of the nucleic acid having a nucleotidesequence of SEQ ID NO:1 to protect murine subjects challenged withHerpes Simplex Virus 2 (HSV-2). The nucleic acid can administered priorto or at the same time as viral challenge.

[0106] The demonstrated ability of these nucleic acids to induce immunestimulation is evidence that the nucleic acids are effective therapeuticagents for vaccination, cancer immunotherapy, asthma immunotherapy,general enhancement of immune function, enhancement of hematopoieticrecovery following radiation or chemotherapy, and other immunemodulatory applications in humans and other subjects.

[0107] The nucleic acids of the invention can be used as stand alonetherapies. A stand alone therapy is a therapy in which aprophylactically or therapeutically beneficial result can be achievedfrom the administration of a single agent or composition. Accordingly,the nucleic acids disclosed herein can be used alone in the preventionor treatment of infectious disease, cancer, and asthma and allergy,because the nucleic acids are capable of inducing immune responses thatare beneficial to the therapeutic outcome of these diseases. Some of themethods described herein relate to the use of nucleic acids as a standalone therapy, while others related to the use of the nucleic acids incombination with other therapeutic agents.

[0108] When used in a vaccine, the nucleic acid is administered with anantigen. Preferably, the antigen is specific for the disorder sought tobe prevented or treated. For example, if the disorder is an infectiousdisease, the antigen is preferably derived from the infectious organism(e.g., bacterium, virus, parasite, fungus, etc.). If the disorder is acancer, the antigen is preferably a cancer antigen.

[0109] The immunostimulatory nucleic acids are useful in some aspects ofthe invention as a prophylactic vaccine for the prevention of aninfection (i.e., an infectious disease), a cancer, an allergy, orasthma. Preferably, prophylactic vaccination is used in subjects thatare not diagnosed with one of these conditions, and more preferably thesubjects are considered at risk of developing one of these conditions.For example, the subject may be one that is at risk of developing aninfection with an infectious organism, or one that is at risk ofdeveloping a cancer in which a specific cancer antigen has beenidentified, or one that is at risk of developing an allergy for which anallergen is known, or one that is at risk of developing asthma where thepredisposition to asthma is known.

[0110] A subject at risk, as used herein, is a subject who has any riskof exposure to an infection causing pathogen, a carcinogen, or anallergen. A subject at risk also includes subjects that have apredisposition to developing such disorders. Some predispositions can begenetic (and can thereby be identified either by genetic analysis or byfamily history). Some predispositions are environmental (e.g., priorexposure to carcinogens, etc.) An example of a subject at risk ofdeveloping an infection is a subject living in or expecting to travel toan area where a particular type of infectious agent is or has beenfound, or it may be a subject who through lifestyle or medicalprocedures is exposed to an organism either directly or indirectly bycontact with bodily fluids that may contain infectious organisms.Subjects at risk of developing infection also include generalpopulations to which a medical agency recommends vaccination for aparticular infectious organism.

[0111] If the antigen is an allergen and the subject develops allergicresponses to that particular antigen and the subject may be exposed tothe antigen, i.e., during pollen season, then that subject is at risk ofexposure to the antigen. A subject at risk of developing an allergy toasthma includes those subjects that have been identified as having anallergy or asthma but that don't have the active disease during theimmunostimulatory nucleic acid treatment as well as subjects that areconsidered to be at risk of developing these diseases because of geneticor environmental factors.

[0112] The immunostimulatory nucleic acids can also be given without theantigen or allergen for shorter term protection against infection,allergy or cancer, and in this case repeated doses will allow longerterm protection.

[0113] A subject at risk of developing a cancer is one who is who has ahigh probability of developing cancer (e.g., a probability that isgreater than the probability within the general public). These subjectsinclude, for instance, subjects having a genetic abnormality, thepresence of which has been demonstrated to have a correlative relationto a likelihood of developing a cancer that is greater than thelikelihood of the general public, and subjects exposed to cancer causingagents (i.e., carcinogens) such as tobacco, asbestos, or other chemicaltoxins, or a subject who has previously been treated for cancer and isin apparent remission. When a subject at risk of developing a cancer istreated with an antigen specific for the type of cancer to which thesubject is at risk of developing and a immunostimulatory nucleic acid,the subject may be able to kill the cancer cells as they develop. If atumor begins to form in the subject, the subject will develop a specificimmune response against the tumor antigen.

[0114] In addition to the use of the immunostimulatory nucleic acids asa prophylactic, the invention also encompasses the use of theimmunostimulatory nucleic acids for the treatment of a subject having aninfection, an allergy, asthma, or a cancer.

[0115] A subject having an infection is a subject that has been exposedto an infectious pathogen and has acute or chronic detectable levels ofthe pathogen in the body, or in bodily waste. When used therapeutically,the immunostimulatory nucleic acids can be used as a stand alone or incombination with another therapeutic agent. For example, theimmunostimulatory nucleic acids can be used therapeutically with anantigen to mount an antigen specific systemic or mucosal immune responsethat is capable of reducing the level of, or eradicating, the infectiouspathogen.

[0116] An infectious disease, as used herein, is a disease arising fromthe presence of a foreign microorganism in the body. It is particularlyimportant to develop effective vaccine strategies and treatments toprotect the body's mucosal surfaces, which are the primary site ofpathogenic entry.

[0117] As used herein, the term treat, treated, or treating when usedwith respect to an infectious disease refers to a prophylactic treatmentwhich increases the resistance of a subject (a subject at risk ofinfection) to infection with a pathogen or, in other words, decreasesthe likelihood that the subject will become infected with the pathogenas well as a treatment after the subject (a subject who has beeninfected) has become infected in order to fight the infection, e.g.,reduce or eliminate the infection or prevent it from becoming worse.

[0118] A subject having an allergy is a subject that has or is at riskof developing an allergic reaction in response to an allergen. Anallergy refers to acquired hypersensitivity to a substance (allergen).Allergic conditions include but are not limited to eczema, allergicrhinitis or coryza, hay fever, conjunctivitis, bronchial asthma,urticaria (hives) and food allergies, and other atopic conditions.

[0119] Currently, allergic diseases are generally treated by theinjection of small doses of antigen followed by subsequent increasingdosage of antigen. It is believed that this procedure inducestolerization to the allergen to prevent further allergic reactions.These methods, however, can take several years to be effective and areassociated with the risk of side effects such as anaphylactic shock. Themethods of the invention avoid these problems.

[0120] Allergies are generally caused by IgE antibody generation againstharmless allergens. The cytokines that are induced by systemic ormucosal administration of immunostimulatory nucleic acids arepredominantly of a class called Th1 (examples are IL-12 and IFN-γ) andthese induce both humoral and cellular immune responses. The types ofantibodies associated with a Th1 response are generally more protectivebecause they have high neutralization and opsonization capabilities. Theother major type of immune response, which is associated with theproduction of IL-4, IL-5 and IL-10 cytokines, is termed a Th2 immuneresponse. Th2 responses involve predominately antibodies and these haveless protective effect against infection and some Th2 isotypes (e.g.,IgE) are associated with allergy. In general, it appears that allergicdiseases are mediated by Th2 type immune responses while Th1 responsesprovide the best protection against infection, although excessive Th1responses are associated with autoimmune disease. Based on the abilityof the immunostimulatory nucleic acids to shift the immune response in asubject from a Th2 (which is associated with production of IgEantibodies and allergy) to a Th1 response (which is protective againstallergic reactions), an effective dose for inducing an immune responseof a immunostimulatory nucleic acid can be administered to a subject totreat or prevent an allergy.

[0121] Thus, the immunostimulatory nucleic acids have significanttherapeutic utility in the treatment of allergic and non-allergicconditions such as asthma. Th2 cytokines, especially IL4 and IL-5 areelevated in the airways of asthmatic subjects. These cytokines promoteimportant aspects of the asthmatic inflammatory response, including IgEisotope switching, eosinophil chemotaxis and activation and mast cellgrowth. Th1 cytokines, especially IFN-γ and IL-12, can suppress theformation of Th2 clones and production of Th2 cytokines. Asthma refersto a disorder of the respiratory system characterized by inflammation,narrowing of the airways and increased reactivity of the airways toinhaled agents. Asthma is frequently, although not exclusivelyassociated with atopic or allergic symptoms.

[0122] A subject having a cancer is a subject that has detectablecancerous cells. The cancer may be a malignant or non-malignant cancer.Cancers or tumors include but are not limited to biliary tract cancer;brain cancer; breast cancer; cervical cancer; choriocarcinoma; coloncancer; endometrial cancer; esophageal cancer; gastric cancer;intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g.small cell and non-small cell); melanoma; neuroblastomas; oral cancer;ovarian cancer; pancreas cancer; prostate cancer; rectal cancer;sarcomas; skin cancer; testicular cancer; thyroid cancer; and renalcancer, as well as other carcinomas and sarcomas. In one embodiment thecancer is hairy cell leukemia, chronic myelogenous leukemia, cutaneousT-cell leukemia, multiple myeloma, follicular lymphoma, malignantmelanoma, squamous cell carcinoma, renal cell carcinoma, prostatecarcinoma, bladder cell carcinoma, or colon carcinoma.

[0123] Some cancer cells are antigenic and thus can be targeted by theimmune system. In one aspect, the combined administration ofimmunostimulatory nucleic acids and cancer medicaments, particularlythose which are classified as cancer immunotherapies, is useful forstimulating a specific immune response against a cancer antigen.

[0124] The theory of immune surveillance is that a prime function of theimmune system is to detect and eliminate neoplastic cells before a tumorforms. A basic principle of this theory is that cancer cells areantigenically different from normal cells and thus elicit immunereactions that are similar to those that cause rejection ofimmunologically incompatible allografts. Studies have confirmed thattumor cells differ, either qualitatively or quantitatively, in theirexpression of antigens. Such antigens are referred to interchangeably astumor antigens or cancer antigens. Some of these antigens may in turn betumor-specific antigens or tumor-associated antigens. “Tumor-specificantigens” are antigens that are specifically present in tumor cells butnot normal cells. Examples of tumor specific antigens are viral antigensin tumors induced by DNA or RNA viruses. “Tumor-associated” antigens arepresent in both tumor cells and normal cells but are present in adifferent quantity or a different form in tumor cells. Examples of suchantigens are oncofetal antigens (e.g., carcinoembryonic antigen),differentiation antigens (e.g., T and Tn antigens), and oncogeneproducts (e.g., HER/neu).

[0125] Different types of cells that can kill tumor targets in vitro andin vivo have been identified: natural killer cells (NK cells), cytolyticT lymphocytes (CTLs), lymphokine-activated killer cells (LAKs), andactivated macrophages. NK cells can kill tumor cells without having beenpreviously sensitized to specific antigens, and the activity does notrequire the presence of class I antigens encoded by the majorhistocompatibility complex (MHC) on target cells. NK cells are thoughtto participate in the control of nascent tumors and in the control ofmetastatic growth. In contrast to NK cells, CTLs can kill tumor cellsonly after they have been sensitized to tumor antigens and when thetarget antigen is expressed on the tumor cells that also express MHCclass I. CTLs are thought to be effector cells in the rejection oftransplanted tumors and of tumors caused by DNA viruses. LAK cells are asubset of null lymphocytes distinct from the NK and CTL populations.Activated macrophages can kill tumor cells in a manner that is notantigen dependent nor MHC restricted once activated. Activatedmacrophages are through to decrease the growth rate of the tumors theyinfiltrate. In vitro assays have identified other immune mechanisms suchas antibody-dependent, cell-mediated cytotoxic reactions and lysis byantibody plus complement. However, these immune effector mechanisms arethought to be less important in vivo than the function of NK, CTLs, LAK,and macrophages in vivo (for review see Piessens, W. F., and David, J.,“Tumor Immunology”, In: Scientific American Medicine, Vol. 2, ScientificAmerican Books, N.Y., pp. 1-13, 1996.

[0126] The goal of immunotherapy is to augment a patient's immuneresponse to an established tumor. One method of immunotherapy includesthe use of adjuvants. Adjuvant substances derived from microorganisms,such as bacillus Calmette-Guerin, heighten the immune response andenhance resistance to tumors in animals.

[0127] An “antigen” as used herein is a molecule capable of provoking animmune response. Antigens include but are not limited to cells, cellextracts, proteins, polypeptides, peptides, polysaccharides,polysaccharide conjugates, peptide and non-peptide mimics ofpolysaccharides and other molecules, small molecules, lipids,glycolipids, carbohydrates, viruses and viral extracts and multicellularorganisms such as parasites and allergens. The term antigen broadlyincludes any type of molecule which is recognized by a host immunesystem as being foreign. Antigens include but are not limited to cancerantigens, microbial antigens, and allergens.

[0128] A “microbial antigen” as used herein is an antigen of amicroorganism and includes but is not limited to virus, bacteria,parasites, and fungi. Such antigens include the intact microorganism aswell as natural isolates and fragments or derivatives thereof and alsosynthetic compounds which are identical to or similar to naturalmicroorganism antigens and induce an immune response specific for thatmicroorganism. A compound is similar to a natural microorganism antigenif it induces an immune response (humoral and/or cellular) to a naturalmicroorganism antigen. Such antigens are used routinely in the art andare well known to those of ordinary skill in the art.

[0129] A “cancer antigen” as used herein is a compound, such as apeptide or protein, present in a tumor or cancer cell and which iscapable of provoking an immune response when expressed on the surface ofan antigen presenting cell in the context of an MHC molecule. Cancerantigens can be prepared from cancer cells either by preparing crudeextracts of cancer cells, for example, as described in Cohen, et al.,1994, Cancer Research, 54:1055, by partially purifying the antigens, byrecombinant technology, or by de novo synthesis of known antigens.Cancer antigens include but are not limited to antigens that arerecombinantly expressed, an immunogenic portion of, or a whole tumor orcancer. Such antigens can be isolated or prepared recombinantly or byany other means known in the art.

[0130] Cancer or tumor antigens are differentially expressed by cancercells and can thereby be exploited in order to target cancer cells. Someof these antigens are encoded, although not necessarily expressed, bynormal cells. These antigens can be characterized as those which arenormally silent (i.e., not expressed) in normal cells, those that areexpressed only at certain stages of differentiation and those that aretemporally expressed such as embryonic and fetal antigens. Other cancerantigens are encoded by mutant cellular genes, such as oncogenes (e.g.,activated ras oncogene), suppressor genes (e.g., mutant p53), fusionproteins resulting from internal deletions or chromosomaltranslocations. Still other cancer antigens can be encoded by viralgenes such as those carried on RNA and DNA tumor viruses.

[0131] In some aspects of the invention, the subject is “exposed to” theantigen. As used herein, the term “exposed to” refers to either theactive step of contacting the subject with an antigen or the passiveexposure of the subject to the antigen in vivo. Methods for the activeexposure of a subject to an antigen are well-known in the art. Ingeneral, an antigen is administered directly to the subject by any meanssuch as intravenous, intramuscular, oral, transdermal, mucosal,intranasal, intratracheal, or subcutaneous administration. The antigencan be administered systemically or locally. Methods for administeringthe antigen and the immunostimulatory nucleic acid are described in moredetail below. A subject is passively exposed to an antigen if an antigenbecomes available for exposure to the immune cells in the body. Asubject may be passively exposed to an antigen, for instance, by entryof a foreign pathogen into the body or by the development of a tumorcell expressing a foreign antigen on its surface.

[0132] Active exposure of the antigen can occur at any time relative tothe administration of the immunostimulatory nucleic acid, includingprior to, simultaneous with, or following nucleic acid administration.In some embodiments, the nucleic acid is administered at the same time,or substantially the same time (e.g., within an hour) of exposure to theantigen, but is administered in a different formulation. As an example,the antigen may be administered locally, and the nucleic acid may beadministered systemically, or vice versa.

[0133] The methods in which a subject is passively exposed to an antigencan be particularly dependent on timing of administration of theimmunostimulatory nucleic acid. For instance, in a subject at risk ofdeveloping a cancer or an infectious disease or an allergic or asthmaticresponse, the subject may be administered the immunostimulatory nucleicacid on a regular basis when that risk is greatest, i.e., during allergyseason or after exposure to a cancer causing agent. Additionally theimmunostimulatory nucleic acid may be administered to travelers beforethey travel to foreign lands where they are at risk of exposure toinfectious agents. Likewise the immunostimulatory nucleic acid may beadministered to soldiers or civilians at risk of exposure to biowarfareto induce a systemic or mucosal immune response to the antigen when andif the subject is exposed to it.

[0134] A subject preferably is a non-rodent subject. A non-rodentsubject shall mean a human or vertebrate animal including but notlimited to a dog, cat, horse, cow, pig, sheep, goat, chicken, primate,e.g., monkey, and fish (aquaculture species), e.g. salmon, butspecifically excluding rodents such as rats and mice.

[0135] Antigens can be derived from various sources including tumor,non-tumor cancers, allergens, and infectious pathogens. Each of thelists recited herein is not intended to be limiting.

[0136] Examples of viruses that have been found in humans include butare not limited to: Retroviridae (e.g. human immunodeficiency viruses,such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, orHIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polioviruses, hepatitis A virus; enteroviruses, human Coxsackie viruses,rhinoviruses, echoviruses); Calciviridae (e.g. strains that causegastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubellaviruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellowfever viruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g.vesicular stomatitis viruses, rabies viruses); Coronaviridae (e.g.coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabiesviruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g.parainfluenza viruses, mumps virus, measles virus, respiratory syncytialvirus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g.Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis Bvirus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses,polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses,vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swinefever virus); and unclassified viruses (e.g. the etiological agents ofSpongiform encephalopathies, the agent of delta hepatitis (thought to bea defective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e. Hepatitis C); Norwalk and related viruses, andastroviruses).

[0137] Although many of the microbial antigens described herein relateto human disorders, the invention is also useful for treating othernon-human vertebrates. Non-human vertebrates are also capable ofdeveloping infections which can be prevented or treated with theimmunostimulatory nucleic acids disclosed herein. For instance, inaddition to the treatment of infectious human diseases, the methods ofthe invention are useful for treating infections of animals.

[0138] Both gram negative and gram positive bacteria serve as antigensin vertebrate animals. Such gram positive bacteria include, but are notlimited to, Pasteurella species, Staphylococci species, andStreptococcus species. Gram negative bacteria include, but are notlimited to, Escherichia coli, Pseudomonas species, and Salmonellaspecies. Specific examples of infectious bacteria include but are notlimited to, Helicobacter pyloris, Borelia burgdorferi, Legionellapneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M.intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcussp., Haemophilus influenzae, Bacillus antracis, corynebacteriumdiphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes,Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp.,Fusobacterium nucleatum, Streptobacillus moniliformis, Treponemapallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomycesisraelli.

[0139] Polypeptides of bacterial pathogens include but are not limitedto an iron-regulated outer membrane protein, (IROMP), an outer membraneprotein (OMP), and an A-protein of Aeromonis salmonicida which causesfurunculosis, p57 protein of Renibacterium salmoninarum which causesbacterial kidney disease (BKD), major surface associated antigen (msa),a surface expressed cytotoxin (mpr), a surface expressed hemolysin(ish), and a flagellar antigen of Yersiniosis; an extracellular protein(ECP), an iron-regulated outer membrane protein (IROMP), and astructural protein of Pasteurellosis; an OMP and a flagellar protein ofVibrosis anguillarum and V. ordalii; a flagellar protein, an OMPprotein, aroA, and purA of Edwardsiellosis ictaluri and E. tarda; andsurface antigen of Ichthyophthirius; and a structural and regulatoryprotein of Cytophaga columnari; and a structural and regulatory proteinof Rickettsia.

[0140] Polypeptides of a parasitic pathogen include but are not limitedto the surface antigens of Ichthyophthirius.

[0141] Examples of fungi include Cryptococcus neoformans, Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydiatrachomatis, Candida albicans. Other infectious organisms (i.e.,protists) include Plasmodium spp. such as Plasmodium falciparum,Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax andToxoplasma gondii. Blood-borne and/or tissues parasites includePlasmodium spp., Babesia microti, Babesia divergens, Leishmania tropica,Leishmania spp., Leishmania braziliensis, Leishmania donovani,Trypanosoma gambiense and Trypanosoma rhodesiense (African sleepingsickness), Trypanosoma cruzi (Chagas' disease), and Toxoplasma gondii.Other medically relevant microorganisms have been described extensivelyin the literature, e.g., see C. G. A Thomas, Medical Microbiology,Bailliere Tindall, Great Britain 1983, the entire contents of which ishereby incorporated by reference.

[0142] Many vaccines for the treatment of non-human vertebrates aredisclosed in Bennett, K. Compendium of Veterinary Products, 3rd ed.North American Compendiums, Inc., 1995. As discussed above, antigensinclude infectious microbes such as virus, parasite, bacteria and fungiand fragments thereof, derived from natural sources or synthetically.Infectious viruses of both human and non-human vertebrates, includeretroviruses, RNA viruses and DNA viruses. This group of retrovirusesincludes both simple retroviruses and complex retroviruses. The simpleretroviruses include the subgroups of B-type retroviruses, C-typeretroviruses and D-type retroviruses. An example of a B-type retrovirusis mouse mammary tumor virus (MMTV). The C-type retroviruses includesubgroups C-type group A (including Rous sarcoma virus (RSV), avianleukemia virus (ALV), and avian myeloblastosis virus (AMV)) and C-typegroup B (including feline leukemia virus (FeLV), gibbon ape leukemiavirus (GALV), spleen necrosis virus (SNV), reticuloendotheliosis virus(RV) and simian sarcoma virus (SSV)). The D-type retroviruses includeMason-Pfizer monkey virus (MPMV) and simian retrovirus type 1 (SRV-1).The complex retroviruses include the subgroups of lentiviruses, T-cellleukemia viruses and the foamy viruses. Lentiviruses include HIV-1, butalso include HIV-2, SIV, Visna virus, feline immunodeficiency virus(FIV), and equine infectious anemia virus (EIAV). The T-cell leukemiaviruses include HTLV-1, HTLV-II, simian T-cell leukemia virus (STLV),and bovine leukemia virus (BLV). The foamy viruses include human foamyvirus (HFV), simian foamy virus (SFV) and bovine foamy virus (BFV).

[0143] Examples of other RNA viruses that are antigens in vertebrateanimals include, but are not limited to, members of the familyReoviridae, including the genus Orthoreovirus (multiple serotypes ofboth mammalian and avian retroviruses), the genus Orbivirus (Bluetonguevirus, Eugenangee virus, Kemerovo virus, African horse sickness virus,and Colorado Tick Fever virus), the genus Rotavirus (human rotavirus,Nebraska calf diarrhea virus, simian rotavirus, bovine or ovinerotavirus, avian rotavirus); the family Picornaviridae, including thegenus Enterovirus (poliovirus, Coxsackie virus A and B, entericcytopathic human orphan (ECHO) viruses, hepatitis A virus, Simianenteroviruses, Murine encephalomyelitis (ME) viruses, Poliovirus muris,Bovine enteroviruses, Porcine enteroviruses , the genus Cardiovirus(Encephalomyocarditis virus (EMC), Mengovirus), the genus Rhinovirus(Human rhinoviruses including at least 113 subtypes; otherrhinoviruses), the genus Apthovirus (Foot and Mouth disease (FMDV); thefamily Calciviridae, including Vesicular exanthema of swine virus, SanMiguel sea lion virus, Feline picornavirus and Norwalk virus; the familyTogaviridae, including the genus Alphavirus (Eastern equine encephalitisvirus, Semliki forest virus, Sindbis virus, Chikungunya virus,O'Nyong-Nyong virus, Ross river virus, Venezuelan equine encephalitisvirus, Western equine encephalitis virus), the genus Flavirius (Mosquitoborne yellow fever virus, Dengue virus, Japanese encephalitis virus, St.Louis encephalitis virus, Murray Valley encephalitis virus, West Nilevirus, Kunjin virus, Central European tick borne virus, Far Eastern tickborne virus, Kyasanur forest virus, Louping III virus, Powassan virus,Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), thegenus Pestivirus (Mucosal disease virus, Hog cholera virus, Borderdisease virus); the family Bunyaviridae, including the genus Bunyvirus(Bunyamwera and related viruses, California encephalitis group viruses),the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fevervirus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus,Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi andrelated viruses); the family Orthomyxoviridae, including the genusInfluenza virus (Influenza virus type A, many human subtypes); Swineinfluenza virus, and Avian and Equine Influenza viruses; influenza typeB (many human subtypes), and influenza type C (possible separate genus);the family paramyxoviridae, including the genus Paramyxovirus(Parainfluenza virus type 1, Sendai virus, Hemadsorption virus,Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumpsvirus), the genus Morbillivirus (Measles virus, subacute sclerosingpanencephalitis virus, distemper virus, Rinderpest virus), the genusPneumovirus (respiratory syncytial virus (RSV), Bovine respiratorysyncytial virus and Pneumonia virus); the family Rhabdoviridae,including the genus Vesiculovirus (VSV), Chandipura virus, Flanders-HartPark virus), the genus Lyssavirus (Rabies virus), fish Rhabdoviruses,and two probable Rhabdoviruses (Marburg virus and Ebola virus); thefamily Arenaviridae, including Lymphocytic choriomeningitis virus (LCM),Tacaribe virus complex, and Lassa virus; the family Coronoaviridae,including Infectious Bronchitis Virus (IBV), Hepatitis virus, Humanenteric corona virus, and Feline infectious peritonitis (Felinecoronavirus).

[0144] Illustrative DNA viruses that are antigens in vertebrate animalsinclude, but are not limited to, the family Poxviridae, including thegenus Orthopoxvirus (Variola major, Variola minor, Monkey pox Vaccinia,Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus(Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avianpoxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genusSuipoxvirus (Swinepox), the genus Parapoxvirus (contagious postulardermatitis virus, pseudocowpox, bovine papular stomatitis virus); thefamily Iridoviridae (African swine fever virus, Frog viruses 2 and 3,Lymphocystis virus of fish); the family Herpesviridae, including thealpha-Herpesviruses (Herpes Simplex Types 1 and 2, Varicella-Zoster,Equine abortion virus, Equine herpes virus 2 and 3, pseudorabies virus,infectious bovine keratoconjunctivitis virus, infectious bovinerhinotracheitis virus, feline rhinotracheitis virus, infectiouslaryngotracheitis virus) the Beta-herpesviruses (Human cytomegalovirusand cytomegaloviruses of swine and monkeys); the gamma-herpesviruses(Epstein-Barr virus (EBV), Marek's disease virus, Herpes saimiri,Herpesvirus ateles, Herpesvirus sylvilagus, guinea pig herpes virus,Lucke tumor virus); the family Adenoviridae, including the genusMastadenovirus (Human subgroups A,B,C,D,E and ungrouped; simianadenoviruses (at least 23 serotypes), infectious canine hepatitis, andadenoviruses of cattle, pigs, sheep, frogs and many other species, thegenus Aviadenovirus (Avian adenoviruses); and non-cultivatableadenoviruses; the family Papoviridae, including the genus Papillomavirus(Human papilloma viruses, bovine papilloma viruses, Shope rabbitpapilloma virus, and various pathogenic papilloma viruses of otherspecies), the genus Polyomavirus (polyomavirus, Simian vacuolating agent(SV40), Rabbit vacuolating agent (RKV), K virus, BK virus, JC virus, andother primate polyoma viruses such as Lymphotrophic papilloma virus);the family Parvoviridae including the genus Adeno-associated viruses,the genus Parvovirus (Feline panleukopenia virus, bovine parvovirus,canine parvovirus, Aleutian mink disease virus, etc). Finally, DNAviruses may include viruses which do not fit into the above familiessuch as Kuru and Creutzfeldt-Jacob disease viruses and chronicinfectious neuropathic agents (CHINA virus).

[0145] The immunostimulatory nucleic acids can also be used to induce animmune response, such as an antigen specific immune response, birds suchas hens, chickens, turkeys, ducks, geese, quail, and pheasant. Birds areprime targets for many types of infections.

[0146] Hatching birds are exposed to pathogenic microorganisms shortlyafter birth. Although these birds are initially protected againstpathogens by maternal derived antibodies, this protection is onlytemporary, and the bird's own immature immune system must begin toprotect the bird against the pathogens. It is often desirable to preventinfection in young birds when they are most susceptible. It is alsodesirable to prevent against infection in older birds, especially whenthe birds are housed in closed quarters, leading to the rapid spread ofdisease. Thus, it is desirable to administer the Immunostimulatorynucleic acid and the non-nucleic acid adjuvant of the invention to birdsto enhance an antigen-specific immune response when antigen is present.

[0147] An example of a common infection in chickens is chickeninfectious anemia virus (CIAV). CIAV was first isolated in Japan in 1979during an investigation of a Marek's disease vaccination break (Yuasa etal., 1979, Avian Dis. 23:366-385). Since that time, CIAV has beendetected in commercial poultry in all major poultry producing countries(van Bulow et al., 1991, pp.690-699) in Diseases of Poultry, 9thedition, Iowa State University Press).

[0148] CIAV infection results in a clinical disease, characterized byanemia, hemorrhage and immunosuppression, in young susceptible chickens.Atrophy of the thymus and of the bone marrow and consistent lesions ofCIAV-infected chickens are also characteristic of CIAV infection.Lymphocyte depletion in the thymus, and occasionally in the bursa ofFabricius, results in immunosuppression and increased susceptibility tosecondary viral, bacterial, or fungal infections which then complicatethe course of the disease. The immunosuppression may cause aggravateddisease after infection with one or more of Marek's disease virus (MDV),infectious bursal disease virus, reticuloendotheliosis virus,adenovirus, or reovirus. It has been reported that pathogenesis of MDVis enhanced by CIAV (DeBoer et al., 1989, p. 28 In Proceedings of the38th Western Poultry Diseases Conference, Tempe, Ariz.). Further, it hasbeen reported that CIAV aggravates the signs of infectious bursaldisease (Rosenberger et al., 1989, Avian Dis. 33:707-713). Chickensdevelop an age resistance to experimentally induced disease due to CAA.This is essentially complete by the age of 2 weeks, but older birds arestill susceptible to infection (Yuasa, N. et al., 1979 supra; Yuasa, N.et al., Arian Diseases 24, 202-209, 1980). However, if chickens aredually infected with CAA and an immunosuppressive agent (IBDV, MDVetc.), age resistance against the disease is delayed (Yuasa, N. et al.,1979 and 1980 supra; Bulow von V. et al., J. Veterinary Medicine 33,93-116, 1986). Characteristics of CIAV that may potentiate diseasetransmission include high resistance to environmental inactivation andsome common disinfectants. The economic impact of CIAV infection on thepoultry industry is clear from the fact that 10% to 30% of infectedbirds in disease outbreaks die.

[0149] Vaccination of birds, like other vertebrate animals can beperformed at any age. Normally, vaccinations are performed at up to 12weeks of age for a live microorganism and between 14-18 weeks for aninactivated microorganism or other type of vaccine. For in ovovaccination, vaccination can be performed in the last quarter of embryodevelopment. The vaccine may be administered subcutaneously, by spray,orally, intraocularly, intratracheally, nasally, or by other mucosaldelivery methods described herein. Thus, the immunostimulatory nucleicacids of the invention can be administered to birds and other non-humanvertebrates using routine vaccination schedules and the antigen can beadministered after an appropriate time period as described herein.Cattle and livestock are also susceptible to infection. Diseases whichaffect these animals can produce severe economic losses, especiallyamongst cattle. The methods of the invention can be used to protectagainst infection in livestock, such as cows, horses, pigs, sheep, andgoats.

[0150] Cows can be infected by bovine viruses. Bovine viral diarrheavirus (BVDV) is a small enveloped positive-stranded RNA virus and isclassified, along with hog cholera virus (HOCV) and sheep border diseasevirus (BDV), in the pestivirus genus. Although, Pestiviruses werepreviously classified in the Togaviridae family, some studies havesuggested their reclassification within the Flaviviridae family alongwith the flavivirus and hepatitis C virus (HCV) groups (Francki, et al.,1991).

[0151] BVDV, which is an important pathogen of cattle can bedistinguished, based on cell culture analysis, into cytopathogenic (CP)and noncytopathogenic (NCP) biotypes. The NCP biotype is more widespreadalthough both biotypes can be found in cattle. If a pregnant cow becomesinfected with an NCP strain, the cow can give birth to a persistentlyinfected and specifically immunotolerant calf that will spread virusduring its lifetime. The persistently infected cattle can succumb tomucosal disease and both biotypes can then be isolated from the animal.Clinical manifestations can include abortion, teratogenesis, andrespiratory problems, mucosal disease and mild diarrhea. In addition,severe thrombocytopenia, associated with herd epidemics, that may resultin the death of the animal has been described and strains associatedwith this disease seem more virulent than the classical BVDVs.

[0152] Equine herpes viruses (EHV) comprise a group of antigenicallydistinct biological agents which cause a variety of infections in horsesranging from subclinical to fatal disease. These include Equineherpesvirus-1 (EHV-1), a ubiquitous pathogen in horses. EHV-1 isassociated with epidemics of abortion, respiratory tract disease, andcentral nervous system disorders. Primary infection of upper respiratorytract of young horses results in a febrile illness which lasts for 8 to10 days. Immunologically experienced mares may be re-infected via therespiratory tract without disease becoming apparent, so that abortionusually occurs without warning. The neurological syndrome is associatedwith respiratory disease or abortion and can affect animals of eithersex at any age, leading to lack of co-ordination, weakness and posteriorparalysis (Telford, E. A. R. et al., Virology 189, 304-316, 1992). OtherEHV's include EHV-2, or equine cytomegalovirus, EHV-3, equine coitalexanthema virus, and EHV-4, previously classified as EHV-1 subtype 2.

[0153] Sheep and goats can be infected by a variety of dangerousmicroorganisms including visna-maedi.

[0154] Primates such as monkeys, apes and macaques can be infected bysimian immunodeficiency virus. Inactivated cell-virus and cell-freewhole simian immunodeficiency vaccines have been reported to affordprotection in macaques (Stott et al. (1990) Lancet 36:1538-1541;Desrosiers et al. PNAS USA (1989) 86:6353-6357; Murphey-Corb et al.(1989) Science 246:1293-1297; and Carlson et al. (1990) AIDS Res. HumanRetroviruses 6:1239-1246). A recombinant HIV gp120 vaccine has beenreported to afford protection in chimpanzees (Berman et al. (1990)Nature 345:622-625).

[0155] Cats, both domestic and wild, are susceptible to infection with avariety of microorganisms. For instance, feline infectious peritonitisis a disease which occurs in both domestic and wild cats, such as lions,leopards, cheetahs, and jaguars. When it is desirable to preventinfection with this and other types of pathogenic organisms in cats, themethods of the invention can be used to vaccinate cats to protect themagainst infection.

[0156] Domestic cats may become infected with several retroviruses,including but not limited to feline leukemia virus (FeLV), felinesarcoma virus (FeSV), endogenous type Concornavirus (RD-114), and felinesyncytia-forming virus (FeSFV). Of these, FeLV is the most significantpathogen, causing diverse symptoms, including lymphoreticular andmyeloid neoplasms, anemias, immune mediated disorders, and animmunodeficiency syndrome which is similar to human acquired immunedeficiency syndrome (AIDS). Recently, a particular replication-defectiveFeLV mutant, designated FeLV-AIDS, has been more particularly associatedwith immunosuppressive properties.

[0157] The discovery of feline T-lymphotropic lentivirus (also referredto as feline immunodeficiency) was first reported in Pedersen et al.(1987) Science 235:790-793. Characteristics of FIV have been reported inYamamoto et al. (1988) Leukemia, December Supplement 2:204S-215S;Yamamoto et al. (1988) Am. J. Vet. Res. 49:1246-1258; and Ackley et al.(1990) J. Virol. 64:5652-5655. Cloning and sequence analysis of FIV havebeen reported in Olmsted et al. (1989) Proc. Natl. Acad. Sci. USA86:2448-2452 and 86:4355-4360.

[0158] Feline infectious peritonitis (FIP) is a sporadic diseaseoccurring unpredictably in domestic and wild Felidae. While FIP isprimarily a disease of domestic cats, it has been diagnosed in lions,mountain lions, leopards, cheetahs, and the jaguar. Smaller wild catsthat have been afflicted with FIP include the lynx and caracal, sandcat, and pallas cat. In domestic cats, the disease occurs predominantlyin young animals, although cats of all ages are susceptible. A peakincidence occurs between 6 and 12 months of age. A decline in incidenceis noted from 5 to 13 years of age, followed by an increased incidencein cats 14 to 15 years old.

[0159] Viral, bacterial, and parasitic diseases in fin-fish, shellfishor other aquatic life forms pose a serious problem for the aquacultureindustry. Owing to the high density of animals in the hatchery tanks orenclosed marine farming areas, infectious diseases may eradicate a largeproportion of the stock in, for example, a fin-fish, shellfish, or otheraquatic life forms facility. Prevention of disease is a more desiredremedy to these threats to fish than intervention once the disease is inprogress. Vaccination of fish is the only preventative method which mayoffer long-term protection through immunity. Nucleic acid basedvaccinations are described in U.S. Pat. No. 5,780,448 issued to Davis.

[0160] The fish immune system has many features similar to the mammalianimmune system, such as the presence of B cells, T cells, lymphokines,complement, and immunoglobulins. Fish have lymphocyte subclasses withroles that appear similar in many respects to those of the B and T cellsof mammals. Vaccines can be administered by immersion or orally.

[0161] Aquaculture species include but are not limited to fin-fish,shellfish, and other aquatic animals. Fin-fish include all vertebratefish, which may be bony or cartilaginous fish, such as, for example,salmonids, carp, catfish, yellowtail, seabream, and seabass. Salmonidsare a family of fin-fish which include trout (including rainbow trout),salmon, and Arctic char. Examples of shellfish include, but are notlimited to, clams, lobster, shrimp, crab, and oysters. Other culturedaquatic animals include, but are not limited to eels, squid, and octopi.

[0162] Polypeptides of viral aquaculture pathogens include but are notlimited to glycoprotein (G) or nucleoprotein (N) of viral hemorrhagicsepticemia virus (VHSV); G or N proteins of infectious hematopoieticnecrosis virus (IHNV); VP1, VP2, VP3 or N structural proteins ofinfectious pancreatic necrosis virus (IPNV); G protein of spring viremiaof carp (SVC); and a membrane-associated protein, tegumin or capsidprotein or glycoprotein of channel catfish virus (CCV).

[0163] Typical parasites infecting horses are Gasterophilus spp.;Eimeria leuckarti, Giardia spp.; Tritrichomonas equi; Babesia spp.(RBC's), Theileria equi; Trypanosoma spp.; Klossiella equi; Sarcocystisspp. Typical parasites infecting swine include Eimeria bebliecki,Eimeria scabra, Isospora suis, Giardia spp.; Balantidium coli, Entamoebahistolytica; Toxoplasma gondii and Sarcocystis spp., and Trichinellaspiralis.

[0164] The major parasites of dairy and beef cattle include Eimeriaspp., Cryptosporidium sp., Giardia spp.; Toxoplasma gondii; Babesiabovis (RBC), Babesia bigemina (RBC), Trypanosoma spp. (plasma),Theileria spp. (RBC); Theileria parva (lymphocytes); Tritrichomonasfoetus; and Sarcocystis spp.

[0165] The major parasites of raptors include Trichomonas gallinae;Coccidia (Eimeria spp.); Plasmodium relictum, Leucocytozoon danilewskyi(owls), Haemoproteus spp., Trypanosoma spp.; Histomonas; Cryptosporidiummeleagridis, Cryptosporidium baileyi, Giardia, Eimeria; Toxoplasma.

[0166] Typical parasites infecting sheep and goats include Eimeria spp.,Cryptosporidium sp., Giardia sp.; Toxoplasma gondii; Babesia spp. (RBC),Trypanosoma spp. (plasma), Theileria spp. (RBC); and Sarcocystis spp.

[0167] Typical parasitic infections in poultry include coccidiosiscaused by Eimeria acervulina, E. necatrix, E. tenella, Isospora spp. andEimeria truncata; histomoniasis, caused by Histomonas meleagridis andHistomonas gallinarum; trichomoniasis caused by Trichomonas gallinae;and hexamitiasis caused by Hexamita meleagridis. Poultry can also beinfected Emeria maxima, Emeria meleagridis, Eimeria adenoeides, Eimeriameleagrimitis, Cryptosporidium, Eimeria brunetti, Emeria adenoeides,Leucocytozoon spp., Plasmodium spp., Hemoproteus meleagridis, Toxoplasmagondii and Sarcocystis.

[0168] The methods of the invention can also be applied to the treatmentand/or prevention of parasitic infection in dogs, cats, birds, fish andferrets. Typical parasites of birds include Trichomonas gallinae;Eimeria spp., Isospora spp., Giardia; Cryptosporidium; Sarcocystis spp.,Toxoplasma gondii, Haemoproteus/Parahaemoproteus, Plasmodium spp.,Leucocytozoon/Akiba, Atoxoplasma, Trypanosoma spp. Typical parasitesinfecting dogs include Trichinella spiralis; Isopora spp., Sarcocystisspp., Cryptosporidium spp., Hammondia spp., Giardia duodenalis (canis);Balantidium coli, Entamoeba histolytica; Hepatozoon canis; Toxoplasmagondii, Trypanosoma cruzi; Babesia canis; Leishmania amastigotes;Neospora caninum.

[0169] Typical parasites infecting feline species include Isospora spp.,Toxoplasma gondii, Sarcocystis spp., Hammondia hammondi, Besnoitia spp.,Giardia spp.; Entamoeba histolytica; Hepatozoon canis, Cytauxzoon sp.,Cytauxzoon sp., Cytauxzoon sp. (red cells, RE cells).

[0170] Typical parasites infecting fish include Hexamita spp., Eimeriaspp.; Cryptobia spp., Nosema spp., Myxosoma spp., Chilodonella spp.,Trichodina spp.; Plistophora spp., Myxosoma Henneguya; Costia spp.,Ichthyophithirius spp., and Oodinium spp.

[0171] Typical parasites of wild mammals include Giardia spp.(carnivores, herbivores), Isospora spp. (carnivores), Eimeria spp.(carnivores, herbivores); Theileria spp. (herbivores), Babesia spp.(carnivores, herbivores), Trypanosoma spp. (carnivores, herbivores);Schistosoma spp. (herbivores); Fasciola hepatica (herbivores),Fascioloides magna (herbivores), Fasciola gigantica (herbivores),Trichinella spiralis (carnivores, herbivores).

[0172] Parasitic infections in zoos can also pose serious problems.Typical parasites of the bovidae family (blesbok, antelope, banteng,eland, gaur, impala, klipspringer, kudu, gazelle) include Eimeria spp.Typical parasites in the pinnipedae family (seal, sea lion) includeEimeria phocae. Typical parasites in the camelidae family (camels,llamas) include Eimeria spp. Typical parasites of the giraffidae family(giraffes) include Eimeria spp. Typical parasites in the elephantidaefamily (African and Asian) include Fasciola spp. Typical parasites oflower primates (chimpanzees, orangutans, apes, baboons, macaques,monkeys) include Giardia sp.; Balantidium coli, Entamoeba histolytica,Sarcocystis spp., Toxoplasma gondii; Plasmodim spp. (RBC), Babesia spp.(RBC), Trypanosoma spp. (plasma), Leishmania spp. (macrophages).

[0173] Cancer is one of the leading causes of death in companion animals(i.e., cats and dogs). Cancer usually strikes older animals which, inthe case of house pets, have become integrated into the family.Forty-five % of dogs older than 10 years of age, are likely to succumbto the disease. The most common treatment options include surgery,chemotherapy and radiation therapy. Others treatment modalities whichhave been used with some success are laser therapy, cryotherapy,hyperthermia and immunotherapy. The choice of treatment depends on typeof cancer and degree of dissemination. Unless the malignant growth isconfined to a discrete area in the body, it is difficult to remove onlymalignant tissue without also affecting normal cells.

[0174] Malignant disorders commonly diagnosed in dogs and cats includebut are not limited to lymphosarcoma, osteosarcoma, mammary tumors,mastocytoma, brain tumor, melanoma, adenosquamous carcinoma, carcinoidlung tumor, bronchial gland tumor, bronchiolar adenocarcinoma, fibroma,myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma,retinoblastoma, Ewing's sarcoma, Wilm's tumor, Burkitt's lymphoma,microglioma, neuroblastoma, osteoclastoma, oral neoplasia, fibrosarcoma,osteosarcoma and rhabdomyosarcoma. Other neoplasias in dogs includegenital squamous cell carcinoma, transmissable venereal tumor,testicular tumor, seminoma, Sertoli cell tumor, hemangiopericytoma,histiocytoma, chloroma (granulocytic sarcoma), corneal papilloma,corneal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma,basal cell tumor, thymoma, stomach tumor, adrenal gland carcinoma, oralpapillomatosis, hemangioendothelioma and cystadenoma. Additionalmalignancies diagnosed in cats include follicular lymphoma, intestinallymphosarcoma, fibrosarcoma and pulmonary squamous cell carcinoma. Theferret, an ever-more popular house pet is known to develop insulinoma,lymphoma, sarcoma, neuroma, pancreatic islet cell tumor, gastric MALTlymphoma and gastric adenocarcinoma.

[0175] Neoplasias affecting agricultural livestock include leukemia,hemangiopericytoma and bovine ocular neoplasia (in cattle); preputialfibrosarcoma, ulcerative squamous cell carcinoma, preputial carcinoma,connective tissue neoplasia and mastocytoma (in horses); hepatocellularcarcinoma (in swine); lymphoma and pulmonary adenomatosis (in sheep);pulmonary sarcoma, lymphoma, Rous sarcoma, reticulendotheliosis,fibrosarcoma, nephroblastoma, B-cell lymphoma and lymphoid leukosis (inavian species); retinoblastoma, hepatic neoplasia, lymphosarcoma(lymphoblastic lymphoma), plasmacytoid leukemia and swimbladder sarcoma(in fish), caseous lumphadenitis (CLA): chronic, infectious, contagiousdisease of sheep and goats caused by the bacterium Corynebacteriumpseudotuberculosis, and contagious lung tumor of sheep caused byjaagsiekte.

[0176] An allergen refers to a substance (antigen) that can induce anallergic or asthmatic response in a susceptible subject. The list ofallergens is enormous and can include pollens, insect venoms, animaldander dust, fungal spores and drugs (e.g. penicillin). Examples ofnatural, animal and plant allergens include but are not limited toproteins specific to the following genuses: Canine (Canis familiaris);Dermatophagoides (e.g. Dermatophagoides farinae); Felis (Felisdomesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g. Loliumperenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica);Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinoasa);Betula (Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa);Artemisia (Artemisia vulgaris); Plantago (e.g. Plantago lanceolata);Parietaria (e.g. Parietaria officinalis or Parietaria judaica);Blattella (e.g. Blattella germanica); Apis (e.g. Apis multiflorum);Cupressus (e.g. Cupressus sempervirens, Cupressus arizonica andCupressus macrocarpa); Juniperus (e.g. Juniperus sabinoides, Juniperusvirginiana, Juniperus communis and Juniperus ashei); Thuya (e.g. Thuyaorientalis); Chamaecyparis (e.g. Chamaecyparis obtusa); Periplaneta(e.g. Periplaneta americana); Agropyron (e.g. Agropyron repens); Secale(e.g. Secale cereale); Triticum (e.g. Triticum aestivum); Dactylis (e.g.Dactylis glomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poapratensis or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g.Holcus lanatus); Anthoxanthum (e.g. Anthoxanthum odoratum);Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostisalba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalarisarundinacea); Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghumhalepensis); and Bromus (e.g. Bromus inermis).

[0177] The antigen may be an antigen that is encoded by a nucleic acidvector or it may be not encoded in a nucleic acid vector. In the formercase the nucleic acid vector is administered to the subject and theantigen is expressed in vivo. In the latter case the antigen may beadministered directly to the subject. An antigen not encoded in anucleic acid vector as used herein refers to any type of antigen that isnot a nucleic acid. For instance, in some aspects of the invention theantigen not encoded in a nucleic acid vector is a polypeptide. Minormodifications of the primary amino acid sequences of polypeptideantigens may also result in a polypeptide which has substantiallyequivalent antigenic activity as compared to the unmodified counterpartpolypeptide. Such modifications may be deliberate, as by site-directedmutagenesis, or may be spontaneous. All of the polypeptides produced bythese modifications are included herein as long as antigenicity stillexists. The polypeptide may be, for example, a viral polypeptide.

[0178] The term “substantially purified” as used herein refers to apolypeptide which is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.One skilled in the art can purify viral or bacterial polypeptides usingstandard techniques for protein purification. The substantially purepolypeptide will often yield a single major band on a non-reducingpolyacrylamide gel. In the case of partially glycosylated polypeptidesor those that have several start codons, there may be several bands on anon-reducing polyacrylamide gel, but these will form a distinctivepattern for that polypeptide. The purity of the viral or bacterialpolypeptide can also be determined by amino-terminal amino acid sequenceanalysis. Other types of antigens not encoded by a nucleic acid vectorsuch as polysaccharides, small molecule, mimics etc are described above,and included within the invention.

[0179] The invention also utilizes polynucleotides encoding theantigenic polypeptides. It is envisioned that the antigen may bedelivered to the subject in a nucleic acid molecule which encodes forthe antigen such that the antigen must be expressed in vivo. Suchantigens delivered to the subject in a nucleic acid vector are referredto as antigens encoded by a nucleic acid vector. The nucleic acidencoding the antigen is operatively linked to a gene expression sequencewhich directs the expression of the antigen nucleic acid within aeukaryotic cell. The gene expression sequence is any regulatorynucleotide sequence, such as a promoter sequence or promoter-enhancercombination, which facilitates the efficient transcription andtranslation of the antigen nucleic acid to which it is operativelylinked.

[0180] The gene expression sequence may, for example, be a mammalian orviral promoter, such as a constitutive or inducible promoter.Constitutive mammalian promoters include, but are not limited to, thepromoters for the following genes: hypoxanthine phosphoribosyltransferase (HPTR), adenosine deaminase, pyruvate kinase, b-actinpromoter and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40),papilloma virus, adenovirus, human immunodeficiency virus (HIV), Roussarcoma virus, cytomegalovirus, the long terminal repeats (LTR) ofMoloney leukemia virus and other retroviruses, and the thymidine kinasepromoter of herpes simplex virus. Other constitutive promoters are knownto those of ordinary skill in the art. The promoters useful as geneexpression sequences of the invention also include inducible promoters.Inducible promoters are expressed in the presence of an inducing agent.For example, the metallothionein promoter is induced to promotetranscription and translation in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart.

[0181] In general, the gene expression sequence shall include, asnecessary, 5′ non-transcribing and 5′ non-translating sequences involvedwith the initiation of transcription and translation, respectively, suchas a TATA box, capping sequence, CAAT sequence, and the like.Especially, such 5′ non-transcribing sequences will include a promoterregion which includes a promoter sequence for transcriptional control ofthe operably joined antigen nucleic acid. The gene expression sequencesoptionally include enhancer sequences or upstream activator sequences asdesired.

[0182] The antigen nucleic acid is operatively linked to the geneexpression sequence. As used herein, the antigen nucleic acid sequenceand the gene expression sequence are said to be operably linked whenthey are covalently linked in such a way as to place the expression ortranscription and/or translation of the antigen coding sequence underthe influence or control of the gene expression sequence. Two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ gene expression sequence results in the transcription of theantigen sequence and if the nature of the linkage between the two DNAsequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the promoter region todirect the transcription of the antigen sequence, or (3) interfere withthe ability of the corresponding RNA transcript to be translated into aprotein.

[0183] Thus, a gene expression sequence would be operably linked to anantigen nucleic acid sequence if the gene expression sequence werecapable of effecting transcription of that antigen nucleic acid sequencesuch that the resulting transcript is translated into the desiredprotein or polypeptide.

[0184] The antigen nucleic acid of the invention may be delivered to theimmune system alone or in association with a vector. In its broadestsense, a vector is any vehicle capable of facilitating the transfer ofthe antigen nucleic acid to the cells of the immune system so that theantigen can be expressed and presented on the surface of the immunecell. The vector generally transports the nucleic acid to the immunecells with reduced degradation relative to the extent of degradationthat would result in the absence of the vector. The vector optionallyincludes the above-described gene expression sequence to enhanceexpression of the antigen nucleic acid in immune cells. In general, thevectors useful in the invention include, but are not limited to,plasmids, phagemids, viruses, other vehicles derived from viral orbacterial sources that have been manipulated by the insertion orincorporation of the antigen nucleic acid sequences. Viral vectors are apreferred type of vector and include, but are not limited to, nucleicacid sequences from the following viruses: retrovirus, such as Moloneymurine leukemia virus, Harvey murine sarcoma virus, murine mammary tumorvirus, and Rous sarcoma virus; adenovirus, adeno-associated virus;SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papillomaviruses; herpes virus; vaccinia virus; polio virus; and RNA virus suchas a retrovirus. One can readily employ other vectors not named butknown in the art.

[0185] Preferred viral vectors are based on non-cytopathic eukaryoticviruses in which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses, the life cycle ofwhich involves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. Retroviruseshave been approved for human gene therapy trials. Most useful are thoseretroviruses that are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high-efficiency transduction ofgenes in vivo.

[0186] Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell lined withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., Gene Transfer and Expression, A Laboratory Manual W. H.Freeman C. O., New York (1990) and Murry, E. J. Methods in MolecularBiology, vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

[0187] A preferred virus for certain applications is theadeno-associated virus, a double-stranded DNA virus. Theadeno-associated virus can be engineered to be replication-deficient andis capable of infecting a wide range of cell types and species. Itfurther has advantages such as, heat and lipid solvent stability; hightransduction frequencies in cells of diverse lineages, includinghemopoietic cells; and lack of superinfection inhibition thus allowingmultiple series of transductions. Reportedly, the adeno-associated viruscan integrate into human cellular DNA in a site-specific manner, therebyminimizing the possibility of insertional mutagenesis and variability ofinserted gene expression characteristic of retroviral infection. Inaddition, wild-type adeno-associated virus infections have been followedin tissue culture for greater than 100 passages in the absence ofselective pressure, implying that the adeno-associated virus genomicintegration is a relatively stable event. The adeno-associated virus canalso function in an extrachromosomal fashion.

[0188] Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well-known to those of skill inthe art. See e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been found to be particularlyadvantageous for delivering genes to cells in vivo because of theirinability to replicate within and integrate into a host genome. Theseplasmids, however, having a promoter compatible with the host cell, canexpress a peptide from a gene operatively encoded within the plasmid.Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40,and pBlueScript. Other plasmids are well-known to those of ordinaryskill in the art. Additionally, plasmids may be custom designed usingrestriction enzymes and ligation reactions to remove and add specificfragments of DNA.

[0189] It has recently been discovered that gene carrying plasmids canbe delivered to the immune system using bacteria. Modified forms ofbacteria such as Salmonella can be transfected with the plasmid and usedas delivery vehicles. The bacterial delivery vehicles can beadministered to a host subject orally or by other administration means.The bacteria deliver the plasmid to immune cells, e.g. B cells,dendritic cells, likely by passing through the gut barrier. High levelsof immune protection have been established using this methodology. Suchmethods of delivery are useful for the aspects of the inventionutilizing systemic delivery of antigen, Immunostimulatory nucleic acidand/or other therapeutic agent.

[0190] Thus, in addition to being suitable as stand alone agents, theimmunostimulatory nucleic acids are useful, inter alia, as vaccineadjuvants. It was previously established that CpG oligonucleotides areexcellent vaccine adjuvants. In order to identify the bestimmunostimulatory nucleic acids for use as a vaccine adjuvant in humansand other non-rodent animals, in vivo screening of different nucleicacids for this purpose was conducted. Several in vitro assays wereevaluated in mice for their predictive value of adjuvant activity invivo. During the course of this study, an in vitro test that ispredictive of in vivo efficacy was identified. It was discovered, rathersurprisingly, that both B cell and NK cell activation correlatedparticularly well with the ability of an immunostimulatory nucleic acidto enhance an in vivo immune response against an antigen.

[0191] The nucleic acids are also useful for improving survival,differentiation, activation and maturation of dendritic cells. Theimmunostimulatory nucleic acids have the unique capability to promotecell survival, differentiation, activation and maturation of dendriticcells. Dendritic precursor cells isolated from blood by immunomagneticcell sorting develop morphologic and functional characteristics ofdendritic cells during a two day incubation with GM-CSF. Without GM-CSFthese cells undergo apoptosis. The immunostimulatory nucleic acids aresuperior to GM-CSF in promoting survival and differentiation ofdendritic cells (MHC II expression, cell size, granularity). Theimmunostimulatory nucleic acids also induce maturation of dendriticcells. Since dendritic cells form the link between the innate and theacquired immune system, by presenting antigens as well as through theirexpression of pattern recognition receptors which detect microbialmolecules like LPS in their local environment, the ability to activatedendritic cells with immunostimulatory nucleic acids supports the use ofthese immunostimulatory nucleic acid based strategies for in vivo andex-vivo immunotherapy against disorders such as cancer and allergic orinfectious diseases. The immunostimulatory nucleic acids are also usefulfor activating and inducing maturation of dendritic cells.

[0192] Immunostimulatory nucleic acids also increase natural killer celllytic activity and antibody dependent cellular cytotoxicity (ADCC). ADCCcan be performed using a immunostimulatory nucleic acid in combinationwith an antibody specific for a cellular target, such as a cancer cell.When the immunostimulatory nucleic acid is administered to a subject inconjunction with the antibody the subject's immune system is induced tokill the tumor cell. The antibodies useful in the ADCC procedure includeantibodies which interact with a cell in the body. Many such antibodiesspecific for cellular targets have been described in the art and manyare commercially available. Examples of these antibodies are listedbelow among the list of cancer immunotherapies.

[0193] The nucleic acids are also useful for redirecting an immuneresponse from a Th2 immune response to a Th1 immune response.Redirection of an immune response from a Th2 to a Th1 immune responsecan be assessed by measuring the levels of cytokines produced inresponse to the nucleic acid (e.g., by inducing monocytic cells andother cells to produce Th1 cytokines, including IL-12, IFN-γ andGM-CSF). The redirection or rebalance of the immune response from a Th2to a Th1 response is particularly useful for the treatment or preventionof asthma. For instance, an effective amount for treating asthma can bethat amount; useful for redirecting a Th2 type of immune response thatis associated with asthma to a Th1 type of response. Th2 cytokines,especially IL-4 and IL-5 are elevated in the airways of asthmaticsubjects. These cytokines promote important aspects of the asthmaticinflammatory response, including IgE isotype switching, eosinophilchemotaxis and activation and mast cell growth. Th1 cytokines,especially IFN-γ and IL-12, can suppress the formation of Th2 clones andproduction of Th2 cytokines. The immunostimulatory nucleic acids of theinvention cause an increase in Th1 cytokines which helps to rebalancethe immune system, preventing or reducing the adverse effects associatedwith a predominately Th2 immune response.

[0194] The invention also includes a method for inducing antigennon-specific innate immune activation and broad spectrum resistance toinfectious challenge using the immunostimulatory nucleic acids. The termantigen non-specific innate immune activation as used herein refers tothe activation of immune cells other than B cells and for instance caninclude the activation of NK cells, T cells or other immune cells thatcan respond in an antigen independent fashion or some combination ofthese cells. A broad spectrum resistance to infectious challenge isinduced because the immune cells are in active form and are primed torespond to any invading compound or microorganism. The cells do not haveto be specifically primed against a particular antigen. This isparticularly useful in biowarfare, and the other circumstances describedabove such as travelers.

[0195] The nucleic acids of the invention can be used in combinationwith other therapeutic agents including anti-microbial agents,adjuvants, cytokines, anti-cancer therapies, allergy medicaments, asthmamedicaments, and the like.

[0196] The nucleic acids of the invention may be administered to asubject with an anti-microbial agent. An anti-microbial agent, as usedherein, refers to a naturally-occurring or synthetic compound which iscapable of killing or inhibiting infectious microorganisms. The type ofanti-microbial agent useful according to the invention will depend uponthe type of microorganism with which the subject is infected or at riskof becoming infected. Anti-microbial agents include but are not limitedto anti-bacterial agents, anti-viral agents, anti-fungal agents andanti-parasitic agents. Phrases such as “anti-infective agent”,“anti-bacterial agent”, “anti-viral agent”, “anti-fungal agent”,“anti-parasitic agent” and “parasiticide” have well-established meaningsto those of ordinary skill in the art and are defined in standardmedical texts. Briefly, anti-bacterial agents kill or inhibit bacteria,and include antibiotics as well as other synthetic or natural compoundshaving similar functions.

[0197] Antibiotics are low molecular weight molecules which are producedas secondary metabolites by cells, such as microorganisms. In general,antibiotics interfere with one or more bacterial functions or structureswhich are specific for the microorganism and which are not present inhost cells. Anti-viral agents can be isolated from natural sources orsynthesized and are useful for killing or inhibiting viruses.Anti-fungal agents are used to treat superficial fungal infections aswell as opportunistic and primary systemic fungal infections.Anti-parasite agents kill or inhibit parasites.

[0198] Antibacterial agents kill or inhibit the growth or function ofbacteria. A large class of antibacterial agents is antibiotics.Antibiotics, which are effective for killing or inhibiting a wide rangeof bacteria, are referred to as broad spectrum antibiotics. Other typesof antibiotics are predominantly effective against the bacteria of theclass gram-positive or gram-negative. These types of antibiotics arereferred to as narrow spectrum antibiotics.

[0199] Other antibiotics which are effective against a single organismor disease and not against other types of bacteria, are referred to aslimited spectrum antibiotics. Antibacterial agents are sometimesclassified based on their primary mode of action. In general,antibacterial agents are cell wall synthesis inhibitors, cell membraneinhibitors, protein synthesis inhibitors, nucleic acid synthesis orfunctional inhibitors, and competitive inhibitors.

[0200] Anti-bacterial agents useful in the invention include but are notlimited to natural penicillins, semi-synthetic penicillins, clavulanicacid, cephalolsporins, bacitracin, ampicillin, carbenicillin, oxacillin,azlocillin, mezlocillin, piperacillin, methicillin, dicloxacillin,nafcillin, cephalothin, cephapirin, cephalexin, cefamandole, cefaclor,cefazolin, cefuroxine, cefoxitin, cefotaxime, cefsulodin, cefetamet,cefixime, ceftriaxone, cefoperazone, ceftazidine, moxalactam,carbapenems, imipenems, monobactems, euztreonam, vancomycin, polymyxin,amphotericin B, nystatin, imidazoles, clotrimazole, miconazole,ketoconazole, itraconazole, fluconazole, rifampins, ethambutol,tetracyclines, chloramphenicol, macrolides, aminoglycosides,streptomycin, kanamycin, tobramycin, amikacin, gentamicin, tetracycline,minocycline, doxycycline, chlortetracycline, erythromycin,roxithromycin, clarithromycin, oleandomycin, azithromycin,chloramphenicol, quinolones, co-trimoxazole, norfloxacin, ciprofloxacin,enoxacin, nalidixic acid, temafloxacin, sulfonamides, gantrisin, andtrimethoprim; Acedapsone Acetosulfone Sodium; Alamecin; Alexidine;Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; AmifloxacinMesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid;Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; AmpicillinSodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate;Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium;Bacampicillin Hydrochloride; Bacitracin; Bacitracin MethyleneDisalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium;Berythromycin; Betamicin Sulfate; Biapenem; Biniramycin; BiphenamineHydrochloride ; Bispyrithione Magsulfex ; Butikacin; Butirosin Sulfate;Capreomycin Sulfate; Carbadox; Carbenicillin Disodium; CarbenicillinIndanyl Sodium; Carbenicillin Phenyl Sodium; Carbenicillin Potassium;Carumonam Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate;Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium;Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime; CefepimeHydrochloride; Cefetecol; Cefixime; Cefmenoxime Hydrochloride;Cefmetazole; Cefmetazole Sodium; Cefonicid Monosodium; Cefonicid Sodium;Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan; CefotetanDisodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium;Cefpimizole; Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium;Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine;Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium;Ceftriaxone Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil;Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; CephalexinHydrochloride; Cephaloglycin; Cephaloridine; Cephalothin Sodium;Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride; Cetophenicol;Chloramphenicol ; Chloramphenicol Palmitate Chloramphenicol PantothenateComplex ; Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate;Chloroxylenol; Chlortetracycline Bisulfate ; ChlortetracyclineHydrochloride ; Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride;Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin;Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride;Clindamycin Phosphate; Clofazimine ; Cloxacillin Benzathine; CloxacillinSodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin;Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; DapsoneDaptomycin; Demeclocycline; Demeclocycline Hydrochloride; Demecycline;Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin Sodium;Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline;Doxycycline Calcium ; Doxycycline Fosfatex; Doxycycline Hyclate;Droxacin Sodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride;Erythromycin; Erythromycin Acistrate; Erythromycin Estolate;Erythromycin Ethylsuccinate; Erythromycin Gluceptate; ErythromycinLactobionate; Erythromycin Propionate; Erythromycin Stearate; EthambutolHydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine;Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin;Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid;Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin;Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem; Isoconazole;Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin;Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin;Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride;Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; MeclocyclineSulfosalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem;Methacycline; Methacycline Hydrochloride; Methenamine; MethenamineHippurate; Methenamine Mandelate; Methicillin Sodium; Metioprim;Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin;Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; MirincamycinHydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; NalidixateSodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin Palmitate;Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate;Neutramycin; Nifuradene; Nifuraldezone; Nifuratel ; Nifuratrone;Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole;Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium;Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium;Oxolinic Acid; Oxytetracycline; Oxytetracycline Calcium; OxytetracyclineHydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin;Pefloxacin Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin GPotassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V;Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin VPotassium; Pentizidone Sodium; Phenyl Aminosalicylate; PiperacillinSodium; Pirbenicillin Sodium; Piridicillin Sodium; PirlimycinHydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate;Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin; Propikacin;Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate; Quinupristin;Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin;Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin;Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin; RosaramicinButyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate;Rosaramicin Stearate; Rosoxacin; Roxarsone; Roxithromycin; Sancycline;Sanfetrinem Sodium; Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin;Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride;Spiramycin; Stallimycin Hydrochloride; Steffunycin; StreptomycinSulfate; Streptonicozid; Sulfabenz ; Sulfabenzamide; Sulfacetamide;Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium;Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine;Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole;Sulfanilate Zinc; Sulfanitran Sulfasalazine; Sulfasomizole;Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl;Sulfisoxazole Diolamine; Sulfomyxin; Sulopenem; Sultamicillin; SuncillinSodium; Talampicillin Hydrochloride; Teicoplanin; TemafloxacinHydrochloride; Temocillin; Tetracycline; Tetracycline Hydrochloride;Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol;Thiphencillin Potassium; Ticarcillin Cresyl Sodium; TicarcillinDisodium; Ticarcillin Monosodium; Ticlatone; Tiodonium Chloride;Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; TrimethoprimSulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate;Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin; andZorbamycin.

[0201] Antiviral agents are compounds which prevent infection of cellsby viruses or replication of the virus within the cell. There are manyfewer antiviral drugs than antibacterial drugs because the process ofviral replication is so closely related to DNA replication within thehost cell, that non-specific antiviral agents would often be toxic tothe host. There are several stages within the process of viral infectionwhich can be blocked or inhibited by antiviral agents. These stagesinclude, attachment of the virus to the host cell (immunoglobulin orbinding peptides), uncoating of the virus (e.g. amantadine), synthesisor translation of viral mRNA (e.g. interferon), replication of viral RNAor DNA (e.g. nucleoside analogues), maturation of new virus proteins(e.g. protease inhibitors), and budding and release of the virus.

[0202] Nucleotide analogues are synthetic compounds which are similar tonucleotides, but which have an incomplete or abnormal deoxyribose orribose group. Once the nucleotide analogues are in the cell, they arephosphorylated, producing the triphosphate formed which competes withnormal nucleotides for incorporation into the viral DNA or RNA. Once thetriphosphate form of the nucleotide analogue is incorporated into thegrowing nucleic acid chain, it causes irreversible association with theviral polymerase and thus chain termination. Nucleotide analoguesinclude, but are not limited to, acyclovir (used for the treatment ofherpes simplex virus and varicella-zoster virus), gancyclovir (usefulfor the treatment of cytomegalovirus), idoxuridine, ribavirin (usefulfor the treatment of respiratory syncitial virus), dideoxyinosine,dideoxycytidine, and zidovudine (azidothymidine). The interferons arecytokines which are secreted by virus-infected cells as well as immunecells. The interferons function by binding to specific receptors oncells adjacent to the infected cells, causing the change in the cellwhich protects it from infection by the virus. α and β-interferon alsoinduce the expression of Class I and Class II MHC molecules on thesurface of infected cells, resulting in increased antigen presentationfor host immune cell recognition. α and β-interferons are available asrecombinant forms and have been used for the treatment of chronichepatitis B and C infection. At the dosages which are effective foranti-viral therapy, interferons have severe side effects such as fever,malaise and weight loss.

[0203] Immunoglobulin therapy is used for the prevention of viralinfection. Immunoglobulin therapy for viral infections is different thanbacterial infections, because rather than being antigen-specific, theimmunoglobulin therapy functions by binding to extracellular virions andpreventing them from attaching to and entering cells which aresusceptible to the viral infection. The therapy is useful for theprevention of viral infection for the period of time that the antibodiesare present in the host. In general there are two types ofimmunoglobulin therapies, normal immunoglobulin therapy andhyper-immunoglobulin therapy. Normal immune globulin therapy utilizes aantibody product which is prepared from the serum of normal blood donorsand pooled. This pooled product contains low titers of antibody to awide range of human viruses, such as hepatitis A, parvovirus,enterovirus (especially in neonates). Hyper-immune globulin therapyutilizes antibodies which are prepared from the serum of individuals whohave high titers of an antibody to a particular virus. Those antibodiesare then used against a specific virus. Examples of hyper-immuneglobulins include zoster immune globulin (useful for the prevention ofvaricella in immuno-compromised children and neonates), human rabiesimmunoglobulin (useful in the post-exposure prophylaxis of a subjectbitten by a rabid animal), hepatitis B immune globulin (useful in theprevention of hepatitis B virus, especially in a subject exposed to thevirus), and RSV immune globulin (useful in the treatment of respiratorysyncitial virus infections).

[0204] Another type of immunoglobulin therapy is active immunization.This involves the administration of antibodies or antibody fragments toviral surface proteins. Two types of vaccines which are available foractive immunization of hepatitis B include serum-derived hepatitis Bantibodies and recombinant hepatitis B antibodies. Both are preparedfrom HBsAg. The antibodies are administered in three doses to subjectsat high risk of infection with hepatitis B virus, such as health careworkers, sexual partners of chronic carriers, and infants.

[0205] Thus, anti-viral agents useful in the invention include but arenot limited to immunoglobulins, amantadine, interferon, nucleosideanalogues, and protease inhibitors. Specific examples of anti-viralsinclude but are not limited to Acemannan; Acyclovir; Acyclovir Sodium;Adefovir; Alovudine; Alvircept Sudotox; Amantadine Hydrochloride;Aranotin; Arildone; Atevirdine Mesylate; Avridine; Cidofovir;Cipamfylline; Cytarabine Hydrochloride; Delavirdine Mesylate;Desciclovir; Didanosine; Disoxaril; Edoxudine; Enviradene; Enviroxime;Famciclovir; Famotine Hydrochloride; Fiacitabine; Fialuridine;Fosarilate; Foscarnet Sodium; Fosfonet Sodium; Ganciclovir; GanciclovirSodium; Idoxuridine; Kethoxal; Lamivudine; Lobucavir; MemotineHydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir;Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate; SomantadineHydrochloride; Sorivudine; Statolon; Stavudine; Tilorone Hydrochloride;Trifluridine; Valacyclovir Hydrochloride; Vidarabine; VidarabinePhosphate; Vidarabine Sodium Phosphate; Viroxime; Zalcitabine;Zidovudine; and Zinviroxime.

[0206] Anti-fungal agents are useful for the treatment and prevention ofinfective fungi. Anti-fungal agents are sometimes classified by theirmechanism of action. Some anti-fungal agents function as cell wallinhibitors by inhibiting glucose synthase. These include, but are notlimited to, basiungin/ECB. Other anti-fungal agents function bydestabilizing membrane integrity. These include, but are not limited to,immidazoles, such as clotrimazole, sertaconzole, fluconazole,itraconazole, ketoconazole, miconazole, and voriconacole, as well as FK463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292,butenafine, and terbinafine. Other anti-fungal agents function bybreaking down chitin (e.g. chitinase) or immunosuppression (501 cream).Some examples of commercially-available agents are shown in Table 1.TABLE 1 Company Brand Name Generic Name Indication Mechanism of ActionPHARMACIA & PNU 196443 PNU 196443 Anti Fungal n/k UPJOHN Lilly LY 303366Basiungin/ECB Fungal Infections Anti-fungal/cell wall inhibitor, glucosesynthase inhibitor Bayer Canesten Clotrimazole Fungal InfectionsMembrane integrity destabilizer Fujisawa FK 463 FK 463 Fungal InfectionsMembrane integrity destabilizer Mylan Sertaconzaole Sertaconzole FungalInfections Membrane integrity destabilizer Genzyme Chitinase ChitinaseFungal Infections, Systemic Chitin Breakdown Liposome AbelcetAmphotericin B, Fungal Infections, Systemic Membrane integritydestabilizer Liposomal Sequus Amphotec Amphotericin B, FungalInfections, Systemic Membrane integrity destabilizer Liposomal Bayer BAY38-9502 BAY 38-9502 Fungal Infections, Systemic Membrane integritydestabilizer Pfizer Diflucan Fluconazole Fungal Infections, SystemicMembrane integrity destabilizer Johnson & Sporanox Itraconazole FungalInfections, Systemic Membrane integrity destabilizer Johnson SepracorItraconzole (2R, Itraconzole (2R, 4S) Fungal Infections, SystemicMembrane integrity destabilizer 4S) Johnson & Nizoral KetoconazoleFungal Infections, Systemic Membrane integrity destabilizer JohnsonJohnson & Monistat Miconazole Fungal Infections, Systemic Membraneintegrity destabilizer Johnson Merck MK 991 MK 991 Fungal Infections,Systemic Membrane integrity destabilizer Bristol Myers PradimicinPradimicin Fungal Infections, Systemic Membrane integrity destabilizerSq'b Pfizer UK-292, 663 UK-292, 663 Fungal Infections, Systemic Membraneintegrity destabilizer Pfizer Voriconazole Voriconazole FungalInfections, Systemic Membrane integrity destabilizer Mylan 501 Cream 501Cream Inflammatory Fungal Immunosuppression Conditions Mylan MentaxButenafine Nail Fungus Membrane Integrity Destabiliser Schering PloughAnti Fungal Anti Fungal Opportunistic Infections Membrane IntegrityDestabiliser Alza Mycelex Troche Clotrimazole Oral Thrush MembraneIntegrity Stabliser Novartis Lamisil Terbinafine Systemic FungalInfections, Membrane Integrity Destabiliser Onychomycosis

[0207] Thus, the anti-fungal agents useful in the invention include butare not limited to imidazoles, FK 463, amphotericin B, BAY 38-9502, MK991, pradimicin, UK 292, butenafine, chitinase, 501 cream, Acrisorcin;Ambruticin; Amorolfine, Amphotericin B; Azaconazole; Azaserine;Basifungin; Bifonazole; Biphenamine Hydrochloride; BispyrithioneMagsulfex ; Butoconazole Nitrate; Calcium Undecylenate; Candicidin;Carbol-Fuchsin; Chlordantoin; Ciclopirox; Ciclopirox Olamine;Cilofungin; Cisconazole; Clotrimazole; Cuprimyxin; Denofungin;Dipyrithione; Doconazole; Econazole; Econazole Nitrate; Enilconazole;Ethonam Nitrate; Fenticonazole Nitrate; Filipin; Fluconazole;Flucytosine; Fungimycin; Griseofulvin; Hamycin; Isoconazole ;Itraconazole; Kalafungin; Ketoconazole; Lomofungin; Lydimycin;Mepartricin ; Miconazole; Miconazole Nitrate; Monensin; Monensin Sodium; Naftifine Hydrochloride; Neomycin Undecylenate; Nifuratel;Nifurmerone; Nitralamine Hydrochloride; Nystatin; Octanoic Acid;Orconazole Nitrate; Oxiconazole Nitrate; Oxifungin Hydrochloride;Parconazole Hydrochloride; Partricin ; Potassium Iodide ; Proclonol ;Pyrithione Zinc ; Pyrrolnitrin; Rutamycin; Sanguinarium Chloride ;Saperconazole; Scopafungin; Selenium Sulfide ; Sinefungin; SulconazoleNitrate; Terbinafine; Terconazole; Thiram; Ticlatone ; Tioconazole;Tolciclate; Tolindate; Tolnaftate; Triacetin; Triafungin; UndecylenicAcid; Viridofulvin; Zinc Undecylenate; and Zinoconazole Hydrochloride.

[0208] Examples of anti-parasitic agents, also referred to asparasiticides useful for human administration include but are notlimited to albendazole, amphotericin B, benznidazole, bithionol,chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine,diethylcarbamazine, diloxanide furoate, eflornithine, furazolidaone,glucocorticoids, halofantrine, iodoquinol, ivermectin, mebendazole,mefloquine, meglumine antimoniate, melarsoprol, metrifonate,metronidazole, niclosamide, nifurtimox, oxamniquine, paromomycin,pentamidine isethionate, piperazine, praziquantel, primaquine phosphate,proguanil, pyrantel pamoate, pyrimethanmine-sulfonamides,pyrimethanmine-sulfadoxine, quinacrine HCl, quinine sulfate, quinidinegluconate, spiramycin, stibogluconate sodium (sodium antimonygluconate), suramin, tetracycline, doxycycline, thiabendazole,tinidazole, trimethroprim-sulfamethoxazole, and tryparsamide some ofwhich are used alone or in combination with others.

[0209] Parasiticides used in non-human subjects include piperazine,diethylcarbamazine, thiabendazole, fenbendazole, albendazole,oxfendazole, oxibendazole, febantel, levamisole, pyrantel tartrate,pyrantel pamoate, dichlorvos, ivermectin, doramectic, milbemycin oxime,iprinomectin, moxidectin, N-butyl chloride, toluene, hygromycin Bthiacetarsemide sodium, melarsomine, praziquantel, epsiprantel,benzimidazoles such as fenbendazole, albendazole, oxfendazole,clorsulon, albendazole, amprolium; decoquinate, lasalocid, monensinsulfadimethoxine; sulfamethazine, sulfaquinoxaline, metronidazole.

[0210] Parasiticides used in horses include mebendazole, oxfendazole,febantel, pyrantel, dichlorvos, trichlorfon, ivermectin, piperazine; forS. westeri: ivermectin, benzimiddazoles such as thiabendazole,cambendazole, oxibendazole and fenbendazole. Useful parasiticides indogs include milbemycin oxine, ivermectin, pyrantel pamoate and thecombination of ivermectin and pyrantel. The treatment of parasites inswine can include the use of levamisole, piperazine, pyrantel,thiabendazole, dichlorvos and fenbendazole. In sheep and goatsanthelmintic agents include levamisole or ivermectin. Caparsolate hasshown some efficacy in the treatment of D. immitis (heartworm) in cats.

[0211] The immunostimulatory nucleic acids may also be administered inconjunction with an anti-cancer therapy. Anti-cancer therapies includecancer medicaments, radiation and surgical procedures. As used herein, a“cancer medicament” refers to a agent which is administered to a subjectfor the purpose of treating a cancer. As used herein, “treating cancer”includes preventing the development of a cancer, reducing the symptomsof cancer, and/or inhibiting the growth of an established cancer. Inother aspects, the cancer medicament is administered to a subject atrisk of developing a cancer for the purpose of reducing the risk ofdeveloping the cancer. Various types of medicaments for the treatment ofcancer are described herein. For the purpose of this specification,cancer medicaments are classified as chemotherapeutic agents,immunotherapeutic agents, cancer vaccines, hormone therapy, andbiological response modifiers.

[0212] As used herein, a “cancer medicament” refers to an agent which isadministered to a subject for the purpose of treating a cancer. As usedherein, “treating cancer” includes preventing the development of acancer, reducing the symptoms of cancer, and/or inhibiting the growth ofan established cancer. In other aspects, the cancer medicament isadministered to a subject at risk of developing a cancer for the purposeof reducing the risk of developing the cancer. Various types ofmedicaments for the treatment of cancer are lo described herein. For thepurpose of this specification, cancer medicaments are classified aschemotherapeutic agents, immunotherapeutic agents, cancer vaccines,hormone therapy, and biological response modifiers. Additionally, themethods of the invention are intended to embrace the use of more thanone cancer medicament along with the immunostimulatory nucleic acids. Asan example, where appropriate, the immunostimulatory nucleic acids maybe administered with a both a chemotherapeutic agent and animmunotherapeutic agent.

[0213] Alternatively, the cancer medicament may embrace animmunotherapeutic agent and a cancer vaccine, or a chemotherapeuticagent and a cancer vaccine, or a chemotherapeutic agent, animmunotherapeutic agent and a cancer vaccine all administered to onesubject for the purpose of treating a subject having a cancer or at riskof developing a cancer.

[0214] Cancer medicaments function in a variety of ways. Some cancermedicaments work by targeting physiological mechanisms that are specificto tumor cells. Examples include the targeting of specific genes andtheir gene products (i.e., proteins primarily) which are mutated incancers. Such genes include but are not limited to oncogenes (e.g., Ras,Her2, bcl-2), tumor suppressor genes (e.g., EGF, p53, Rb), and cellcycle targets (e.g., CDK4, p21, telomerase). Cancer medicaments canalternately target signal transduction pathways and molecular mechanismswhich are altered in cancer cells. Targeting of cancer cells via theepitopes expressed on their cell surface is accomplished through the useof monoclonal antibodies. This latter type of cancer medicament isgenerally referred to herein as immunotherapy.

[0215] Other cancer medicaments target cells other than cancer cells.For example, some medicaments prime the immune system to attack tumorcells (i.e., cancer vaccines). Still other medicaments, calledangiogenesis inhibitors, function by attacking the blood supply of solidtumors. Since the most malignant cancers are able to metastasize (i.e.,exist the primary tumor site and seed a distal tissue, thereby forming asecondary tumor), medicaments that impede this metastasis are alsouseful in the treatment of cancer. Angiogenic mediators include basicFGF, VEGF, angiopoietins, angiostatin, endostatin, TNFα, TNP-470,thrombospondin-1, platelet factor 4, CAI, and certain members of theintegrin family of proteins. One category of this type of medicament isa metalloproteinase inhibitor, which inhibits the enzymes used by thecancer cells to exist the primary tumor site and extravasate intoanother tissue.

[0216] Immunotherapeutic agents are medicaments which derive fromantibodies or antibody fragments which specifically bind or recognize acancer antigen. As used herein a cancer antigen is broadly defined as anantigen expressed by a cancer cell. Preferably, the antigen is expressedat the cell surface of the cancer cell. Even more preferably, theantigen is one which is not expressed by normal cells, or at least notexpressed to the same level as in cancer cells. Antibody-basedimmunotherapies may function by binding to the cell surface of a cancercell and thereby stimulate the endogenous immune system to attack thecancer cell. Another way in which antibody-based therapy functions is asa delivery system for the specific targeting of toxic substances tocancer cells. Antibodies are usually conjugated to toxins such as ricin(e.g., from castor beans), calicheamicin and maytansinoids, toradioactive isotopes such as Iodine-131 and Yttrium-90, tochemotherapeutic agents (as described herein), or to biological responsemodifiers. In this way, the toxic substances can be concentrated in theregion of the cancer and non-specific toxicity to normal cells can beminimized. In addition to the use of antibodies which are specific forcancer antigens, antibodies which bind to vasculature, such as thosewhich bind to endothelial cells, are also useful in the invention. Thisis because generally solid tumors are dependent upon newly formed bloodvessels to survive, and thus most tumors are capable of recruiting andstimulating the growth of new blood vessels. As a result, one strategyof many cancer medicaments is to attack the blood vessels feeding atumor and/or the connective tissues (or stroma) supporting such bloodvessels.

[0217] The use of immunostimulatory nucleic acids in conjunction withimmunotherapeutic agents such as monoclonal antibodies is able toincrease long-term survival through a number of mechanisms includingsignificant enhancement of ADCC (as discussed above), activation ofnatural killer (NK) cells and an increase in IFNα levels. The nucleicacids when used in combination with monoclonal antibodies serve toreduce the dose of the antibody required to achieve a biological result.

[0218] Examples of cancer immunotherapies which are currently being usedor which are in development are listed in Table 2. TABLE 2 CancerImmunotherapies in Development or on the Market MARKETER BRAND NAME(GENERIC NAME) INDICATION IDEC/Genentech, Rituxan ™ (rituximab,Mabthera) (IDEC- non-Hodgkin's Inc./Hoffmann-LaRoche C2B8, chimericmurine/human anti-CD20 lymphoma (first monoclonal antibody MAb) licensedfor the treatment of cancer in the U.S.) Genentech/Hoffmann-LaHerceptin, anti-Her2 hMAb Breast/ovarian Roche Cytogen Corp. Quadramet(CYT-424) radiotherapeutic Bone metastases agentCentocor/Glaxo/Ajinomoto Panorex ® (17-1A) (murine m onoclonal Adjuvanttherapy for antibody) colorectal (Dukes-C) Centocor/Ajinomoto Panorex ®(17-1A) (chimeric murine Pancreatic, lung, monoclonal antibody) breast,ovary IDEC IDEC-Y2B8 (murine, anti-CD20 MAb non-Hodgkin's labeled withYttrium-90) lymhoma ImClone Systems BEC2 (anti-idiotypic MAb, mimics theSmall cell lung GD₃ epitope) (with BCG) ImClone Systems C225 (chimericmonoclonal antibody to Renal cell epidermal growth factor receptor(EGFr)) Techniclone Oncolym (Lym-1 monoclonal antibody non-Hodgkin'sInternational/Alpha linked to 131 iodine) lymphoma Therapeutics ProteinDesign Labs SMART M195 Ab, humanized Acute myleoid leukemia Techniclone¹³¹I LYM-1 (Oncolym ™) non-Hodgkin's Corporation/Cambridge lymphomaAntibody Technology Aronex Pharmaceuticals, ATRAGEN ® Acutepromyelocytic Inc. leukemia ImClone Systems C225 (chimeric anti-EGFrmonoclonal Head & neck, non- antibody) + cisplatin or radiation smallcell lung cancer Altarex, Canada Ovarex (B43.13, anti-idiotypic CA125,Ovarian mouse MAb) Coulter Pharma (Clinical Bexxar (anti-CD20 Mablabeled with ¹³¹I) non-Hodgkin's results have been positive, lymphomabut the drug has been associated with significant bone marrow toxicity)Aronex Pharmaceuticals, ATRAGEN ® Kaposi's sarcoma Inc. IDECPharmaceuticals Rituxan ™ (MAb against CD20) pan-B Ab B cell lymphomaCorp./Genentech in combo. with chemotherapy LeukoSite/Ilex OncologyLDP-03, huMAb to the leukocyte antigen Chronic lymphocytic CAMPATHleukemia (CLL) Center of Molecular ior t6 (anti CD6, murine MAb) CTCLCancer Immunology Medarex/Novartis MDX-210 (humanized anti-HER-2 Breast,ovarian bispecific antibody) Medarex/Novartis MDX-210 (humanizedanti-HER-2 Prostate, non-small cell bispecific antibody) lung,pancreatic, breast Medarex MDX-11 (complement activating receptor Acutemyelogenous (CAR) monoclonal antibody) leukemia (AML) Medarex/NovartisMDX-210 (humanized anti-HER-2 Renal and colon bispecific antibody)Medarex MDX-11 (complement activating receptor Ex vivo bone marrow (CAR)monoclonal antibody) purging in acute myelogenous leukemia (AML) MedarexMDX-22 (humanized bispecific antibody, Acute myleoid MAb-conjugates)(complement cascade leukemia activators) Cytogen OV103 (Yttrium-90labelled antibody) Ovarian Cytogen OV103 (Yttrium-90 labelled antibody)Prostate Aronex Pharmaceuticals, ATRAGEN ® non-Hodgkin's Inc. lymphomaGlaxo Wellcome plc 3622W94 MAb that binds to EGP40 (17- non-small celllung, 1A) pancarcinoma antigen on prostate (adjuvant) adenocarcinomasGenentech Anti-VEGF, RhuMAb (inhibits Lung, breast, prostate,angiogenesis) colorectal Protein Design Labs Zenapax (SMART Anti-Tac(IL-2 Leukemia, lymphoma receptor) Ab, humanized) Protein Design LabsSMART M195 Ab, humanized Acute promyelocytic leukemia ImClone SystemsC225 (chimeric anti-EGFr monoclonal Breast antibody) + taxol ImCloneSystems (licensed C225 (chimeric anti-EGFr monoclonal prostate from RPR)antibody) + doxorubicin ImClone Systems C225 (chimeric anti-EGFrmonoclonal prostate antibody) + adriamycin ImClone Systems BEC2(anti-idiotypic MAb, mimics the Melanoma GD₃ epitope) Medarex MDX-210(humanized anti-HER-2 Cancer bispecific antibody) Medarex MDX-220(bispecific for tumors that Lung, colon, prostate, express TAG-72)ovarian, endometrial, pancreatic and gastric Medarex/Novartis MDX-210(humanized anti-HER-2 Prostate bispecific antibody) Medarex/Merck KgaAMDX-447 (humanized anti-EGF receptor EGF receptor cancers bispecificantibody) (head & neck, prostate, lung, bladder, cervical, ovarian)Medarex/Novartis MDX-210 (humanized anti-HER-2 Comb. Therapy withbispecific antibody) G-CSF for various cancers, esp. breast IDECMELIMMUNE-2 (murine monoclonal Melanoma antibody therapeutic vaccine)IDEC MELIMMUNE-1 (murine monoclonal Melanoma antibody therapeuticvaccine) Immunomedics, Inc. CEACIDE ™ (I-131) Colorectal and other NeoRxPretarget ™ radioactive antibodies non-Hodgkin's B cell lymphomaNovopharm Biotech, Inc. NovoMAb-G2 (pancarcinoma specific Cancer Ab)Techniclone Corporation/ TNT (chimeric MAb to histone antigens) BrainCambridge Antibody Technology Techniclone International/ TNT (chimericMAb to histone antigens) Brain Cambridge Antibody Technology NovopharmGliomab-H (Monoclonals - Humanized Brain, melanomas, Abs) neuroblastomasGenetics Institute/AHP GNI-250 Mab Colorectal Merck KgaA EMD-72000(chimeric-EGF antagonist) Cancer Immunomedics LymphoCide (humanized LL2antibody) non-Hodgkin's B-cell lymphoma Immunex/AHP CMA 676 (monoclonalantibody Acute myelogenous conjugate) leukemia Novopharm Biotech, Inc.Monopharm-C Colon, lung, pancreatic Novopharm Biotech, Inc. 4B5anti-idiotype Ab Melanoma, small-cell lung Center of Molecular ioregf/r3 (anti EGF-R humanized Ab) Radioimmunotherapy Immunology Center ofMolecular ior c5 (murine MAb colorectal) for Colorectal Immunologyradioimmunotherapy Creative BioMolecules/ BABS (biosynthetic antibodybinding site) Breast cancer Chiron Proteins ImClone Systems/Chugai FLK-2(monoclonal antibody to fetal liver Tumor-associated kinase-2 (FLK-2))angiogenesis ImmunoGen, Inc. Humanized MAb/small-drug conjugateSmall-cell lung Medarex, Inc. MDX-260 bispecific, targets GD-2 Melanoma,glioma, neuroblastoma Procyon Biopharma, Inc. ANA Ab Cancer ProteinDesign Labs SMART 1D10 Ab B-cell lymphoma Protein Design SMART ABL 364Ab Breast, lung, colon Labs/Novartis Immunomedics, Inc. ImmuRAIT-CEAColorectal

[0219] Yet other types of chemotherapeutic agents which can be usedaccording to the invention include Aminoglutethimide, Asparaginase,Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl, Dactinomycin,Daunorubicin HCl, Estramustine phosphate sodium, Etoposide (VP16-213),Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolideacetate (LHRH-releasing factor analogue), Lomustine (CCNU),Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna, Mitotane(o.p{acute over ()}-DDD), Mitoxantrone HCl, Octreotide, Plicamycin,Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine,Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA), Azacitidine,Erthropoietin, Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone(methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Pentostatin(2′deoxycoformycin), Semustine (methyl-CCNU), Teniposide (VM-26) andVindesine sulfate.

[0220] Cancer vaccines are medicaments which are intended to stimulatean endogenous immune response against cancer cells. Currently producedvaccines predominantly activate the humoral immune system (i.e., theantibody dependent immune response). Other vaccines currently indevelopment are focused on activating the cell-mediated immune systemincluding cytotoxic T lymphocytes which are capable of killing tumorcells. Cancer vaccines generally enhance the presentation of cancerantigens to both antigen presenting cells (e.g., macrophages anddendritic cells) and/or to other immune cells such as T cells, B cells,and NK cells.

[0221] Although cancer vaccines may take one of several forms, asdiscussed infra, their purpose is to deliver cancer antigens and/orcancer associated antigens to antigen presenting cells (APC) in order tofacilitate the endogenous processing of such antigens by APC and theultimate presentation of antigen presentation on the cell surface in thecontext of MHC class I molecules. One form of cancer vaccine is a wholecell vaccine which is a preparation of cancer cells which have beenremoved from a subject, treated ex vivo and then reintroduced as wholecells in the subject. Lysates of tumor cells can also be used as cancervaccines to elicit an immune response. Another form cancer vaccine is apeptide vaccine which uses cancer-specific or cancer-associated smallproteins to activate T cells. Cancer-associated proteins are proteinswhich are not exclusively expressed by cancer cells (i.e., other normalcells may still express these antigens). However, the expression ofcancer-associated antigens is generally consistently upregulated withcancers of a particular type. Yet another form of cancer vaccine is adendritic cell vaccine which includes whole dendritic cells which havebeen exposed to a cancer antigen or a cancer-associated antigen invitro. Lysates or membrane fractions of dendritic cells may also be usedas cancer vaccines. Dendritic cell vaccines are able to activateantigen-presenting cells directly. Other cancer vaccines includeganglioside vaccines, heat-shock protein vaccines, viral and bacterialvaccines, and nucleic acid vaccines.

[0222] The use of immunostimulatory nucleic acids in conjunction withcancer vaccines provides an improved antigen-specific humoral and cellmediated immune response, in addition to activating NK cells andendogenous dendritic cells, and increasing IFNα levels. This enhancementallows a vaccine with a reduced antigen dose to be used to achieve thesame beneficial effect. In some instances, cancer vaccines may be usedalong with adjuvants, such as those described above.

[0223] Other vaccines take the form of dendritic cells which have beenexposed to cancer antigens in vitro, have processed the antigens and areable to express the cancer antigens at their cell surface in the contextof MHC molecules for effective antigen presentation to other immunesystem cells.

[0224] The immunostimulatory nucleic acids are used in one aspect of theinvention in conjunction with cancer vaccines which are dendritic cellbased. A dendritic cell is a professional antigen presenting cell.Dendritic cells form the link between the innate and the acquired immunesystem by presenting antigens and through their expression of patternrecognition receptors which detect microbial molecules like LPS in theirlocal environment. Dendritic cells efficiently internalize, process, andpresent soluble specific antigen to which it is exposed. The process ofinternalizing and presenting antigen causes rapid upregulation of theexpression of major histocompatibility complex (MHC) and costimulatorymolecules, the production of cytokines, and migration toward lymphaticorgans where they are believed to be involved in the activation of Tcells.

[0225] Table 3 lists a variety of cancer vaccines which are eithercurrently being used or are in development. TABLE 3 Cancer Vaccines inDevelopment or on the Market MARKETER NAME) INDICATION Center ofMolecular EGF Cancer Immunology Center of Molecular Ganglioside cancerImmunology vaccine Center of Molecular Anti-idiotypic Cancer vaccineImmunology ImClone Systems/Memorial Gp75 antigen MelanomaSloan-Kettering Cancer Center ImClone Systems/Memorial Anti-idiotypicAbs Cancer vaccines Sloan-Kettering Cancer Center ProgenicsPharmaceuticals, Inc. GMK melanoma vaccine Melanoma ProgenicsPharmaceuticals, Inc, MGV ganglioside conjugate Lymphoma, colorectal,vaccine lung Corixa Her2/neu Breast, ovarian AltaRex Ovarex Ovarian AVAXTechnologies Inc. M-Vax, autologous whole cell Melanoma AVAXTechnologies Inc. O-Vax, autologous whole cell Ovarian AVAX TechnologiesInc. L-Vax, autologous whole cell Leukemia-AML Biomira Inc./ChironTheratope, STn-KLH Breast, Colorectal Biomira Inc. BLP25, MUC-1 peptidevaccine Lung encapsulated in liposomal delivery system Biomira Inc.BLP25, MUC-1 peptide vaccine Lung encapsulated in liposomal deliverysystem + Liposomal IL-2 Biomira Inc. Liposomal idiotypic vaccineLymphoma B-cell malignancies Ribi Immunochem Melacine, cell lysateMelanoma Corixa Peptide antigens, microsphere Breast delivery sysem andLeIF adjuvant Corixa Peptide antigens, microsphere Prostate deliverysysem and LeIF adjuvant Corixa Peptide antigens, microsphere Ovariandelivery sysem and LeIF adjuvant Corixa Peptide antigens, microsphereLymphoma delivery sysem and LeIF adjuvant Corixa Peptide antigens,microsphere Lung delivery sysem and LeIF adjuvant Virus ResearchInstitute Toxin/antigen recombinant All cancers delivery system ApollonInc. Genevax-TCR T-cell lymphoma Bavarian Nordic Research MVA-based(vaccinia virus) Melanoma Institute A/S vaccine BioChem Pharma/BioChemPACIS, BCG vaccine Bladder Vaccine Cantab Pharmaceuticals TA-HPVCervical Cantab Pharmaceuticals TA-CIN Cervical Cantab PharmaceuticalsDISC-Virus, immunotherapy Cancer Pasteur Merieux Connaught ImmuCyst ®/TheraCys ® - B CG Bladder Immunotherapeutic (BacillusCalmette-Guerin/Connaught), for intravesical treatment of superficialbladder cancer

[0226] As used herein, chemotherapeutic agents embrace all other formsof cancer medicaments which do not fall into the categories ofimmunotherapeutic agents or cancer vaccines. Chemotherapeutic agents asused herein encompass both chemical and biological agents. These agentsfunction to inhibit a cellular activity which the cancer cell isdependent upon for continued survival. Categories of chemotherapeuticagents include alkylating/alkaloid agents, antimetabolites, hormones orhormone analogs, and miscellaneous antineoplastic drugs. Most if not allof these agents are directly toxic to cancer cells and do not requireimmune stimulation. Combination chemotherapy and immunostimulatorynucleic acid administration increases the maximum tolerable dose ofchemotherapy.

[0227] Chemotherapeutic agents which are currently in development or inuse in a clinical setting are shown in Table 4. TABLE 4 Cancer Drugs inDevelopment or on the Market Marketer Brand Name Generic Name IndicationAbbott TNP 470/AGM 1470 Fragyline Anti-Angiogenesis in Cancer Takeda TNP470/AGM 1470 Fragyline Anti-Angiogenesis in Cancer Scotia Meglamine GLAMeglamine GLA Bladder Cancer Medeva Valstar Valrubicin Bladder Cancer -Refractory in situ carcinoma Medeva Valstar Valrubicin Bladder Cancer -Papillary Cancer Rhone Poulenc Gliadel Wafer Carmustaine + PolifeprBrain Tumor Osan Warner Lambert Undisclosed Cancer (b) UndisclosedCancer (b) Cancer Bristol Myers RAS Famesyl Transferase RASFamesylTransferase Cancer Squib Inhibitor Inhibitor Novartis MMI 270 MMI270 Cancer Bayer BAY 12-9566 BAY 12-9566 Cancer Merck FamesylTransferase Inhibitor Famesyl Transferase Cancer (Solid tumors -Inhibitor pancrease, colon, lung, breast) Pfizer PFE MMP Cancer,angiogenesis Pfizer PFE Tyrosine Kinase Cancer, angiogenesis LillyMTA/LY 231514 MTA/LY 231514 Cancer Solid Tumors Lilly LY264618/Lometexol Lometexol Cancer Solid Tumors Scotia Glamolec LiGLA(lithium-gamma Cancer, pancreatic, breast, linolenate) colon WarnerLambert CI-994 CI-994 Cancer, Solid Tumors/ Leukemia Schering AGAngiogenesis inhibitor Angiogenesis Inhibitor Cancer/Cardio TakedaTNP-470 n/k Malignant Tumor Smithkline Hycamtin Topotecan MetastaticOvarian Cancer Beecham Novartis PKC 412 PKC 412 Multi-Drug ResistantCancer Novartis Valspodar PSC 833 Myeloid Leukemia/Ovarian CancerImmunex Novantrone Mitoxantrone Pain related to hormone refractoryprostate cancer. Warner Lambert Metaret Suramin Prostate GenentechAnti-VEGF Anti-VEGF Prostate/Breast/ Colorectal/NSCL Cancer BritishBiotech Batimastat Batimastat (BB94) Pterygium Eisai E 7070 E 7070 SolidTumors Biochem BCH-4556 BCH-4556 Solid Tumors Pharma Sankyo CS-682CS-682 Solid Tumors Agouron AG2037 AG2037 Solid Tumors IDEC Pharma 9-AC9-AC Solid Tumors Agouron VEGF/b-FGF Inhibitors VEGF/b-FGF InhibitorsSolid Tumors Agouron AG3340 AG3340 Solid Tumors/Macular Degen VertexIncel VX-710 Solid Tumors - IV Vertex VX-853 VX-853 Solid Tumors - OralZeneca ZD 0101 (inj) ZD 0101 Solid Tumors Novartis ISI 641 ISI 641 SolidTumors Novartis ODN 698 ODN 698 Solid Tumors Tanube Seiyaku TA 2516Marimistat Solid Tumors British Biotech Marimastat Marimastat (BB 2516)Solid Tumors Celltech CDP 845 Aggrecanase Inhibitor Solid Tumors/BreastCancer Chiroscience D2163 D2163 Solid Tumors/Metastases Warner LambertPD 183805 PD 183805 Daiichi DX8951f DX8951f Anti-Cancer Daiichi LemonalDP 2202 Lemonal DP 2202 Anti-Cancer Fujisawa FK 317 FK 317 AnticancerAntibiotic Chugai Picibanil OK-432 Antimalignant Tumor Nycomed AD32/valrubicin Valrubicin Bladder Cancer-Refractory Amersham InsituCarcinoma Nycomed Metastron Strontium Derivative Bone Cancer (adjuntAmersham therapy, Pain) Schering Plough Temodal Temozolomide BrainTumours Schering Plough Temodal Temozolonide Brain Tumours LiposomeEvacet Doxorubicin, Liposomal Breast Cancer Nycomed Yewtaxan PaclitaxelBreast Cancer Advanced, Amersham Ovarian Cancer Advanced Bristol MyersTaxol Paclitaxel Breast Cancer Advanced, Squib Ovarian Cancer Advanced,NSCLC Roche Xeloda Capecitabine Breast Cancer, Colorectal Cancer RocheFurtulon Doxifluridine Breast Cancer, Colorectal Cancer, Gastric CancerPharmacia & Adriamycin Doxorubicin Breast Cancer, Leukemia Upjohn IvaxCyclopax Paclitaxel, Oral Breast/Ovarian Cancer Rhone Poulenc OralTaxoid Oral Taxoid Broad Cancer AHP Novantrone Mitoxantrone CancerSequus SPI-077 Cisplatin, Stealth Cancer Hoechst HMR 1275 FlavopiridolCancer Pfizer CP-358, 774 EGFR Cancer Pfizer CP-609, 754 RAS OncogeneInhibitor Cancer Bristol Myers BMS-182751 Oral Platinum Cancer (Lung,Ovarian) Squib Bristol Myers UFT (Tegafur/Uracil) UFT (Tegafur/Uracil)Cancer Oral Squib Johnson & Ergamisol Levamisole Cancer Therapy JohnsonGlaxo Wellcome Eniluracil/776C85 5FU Enhancer Cancer, Refractory Solid &Colorectal Cancer Johnson & Ergamisol Levamisole Colon Cancer JohnsonRhone Poulenc Campto Irinotecan Colorectal Cancer, Cervical CancerPharmacia & Camptosar Irinotecan Colorectal Cancer, Cervical UpjohnCancer Zeneca Tomudex Ralitrexed Colorectal Cancer, Lung Cancer, BreastCancer Johnson & Leustain Cladribine Hairy Cell Leukaemia Johnson IvaxPaxene Paclitaxel Kaposi Sarcoma Sequus Doxil Doxorubicin, LiposomalKS/Cancer Sequus Caelyx Doxorubicin, Liposomal KS/Cancer Schering AGFludara Fludarabine Leukaemia Pharmacia & Pharmorubicin EpirubicinLung/Breast Cancer Upjohn Chiron DepoCyt DepoCyt Neoplastic MeningitisZeneca ZD1839 ZD 1839 Non Small Cell Lung Cancer, Pancreatic Cancer BASFLU 79553 Bis-Naphtalimide Oncology BASF LU 103793 Dolastain OncologyShering Plough Caetyx Doxorubicin-Liposome Ovarian/Breast Cancer LillyGemzar Gemcitabine Pancreatic Cancer, Non Small Cell Lung Cancer,Breast, Bladder and Ovarian Zeneca ZD 0473/Anormed ZD 0473/AnormedPlatinum based NSCL, ovarian etc. Yamanouchi YM 116 YM 116 ProstateCancer Nycomed Seeds/I-125 Rapid St Lodine Seeds Prostate CancerAmersham Agouron Cdk4/cdk2 inhibitors cdk4/cdk2 inhibitors Solid TumorsAgouron PARP inhibitors PARP Inhibitors Solid Tumors Chiroscience D4809Dexifosamide Solid Tumors Bristol Myers UFT (Tegafur/Uracil) UFT(Tegafur/Uracil) Solid Tumors Squib Sankyo Krestin Krestin Solid TumorsAsta Medica Ifex/Mesnex Ifosamide Solid Tumors Bristol MeyersIfex/Mesnex Ifosamide Solid Tumors Squib Bristol Myers Vumon TeniposideSolid Tumors Squib Bristol Myers Paraplatin Carboplatin Solid TumorsSquib Bristol Myers Plantinol Cisplatin, Stealth Solid Tumors SquibBristol Myers Plantinol Cisplatin Solid Tumors Squib Bristol MyersVepeside Etoposide Solid Tumors Melanoma Squib Zeneca ZD 9331 ZD 9331Solid Tumors, Advanced Colorectal Chugai Taxotere Docetaxel SolidTumors, Breast Cancer Rhone Poulenc Taxotere Docetaxel Solid Tumors,Breast Cancer Glaxo Wellcome Prodrug of guanine Prodrug of arabinside TCell Leukemia/Lymphoma arabinside & B Cell Neoplasm Bristol Myers TaxaneAnalog Taxane Analog Taxol follow up Squib

[0228] In one embodiment, the methods of the invention useimmunostimulatory nucleic acids as a replacement to the use of IFNαtherapy in the treatment of cancer. Currently, some treatment protocolscall for the use of IFNα. Since IFNα is produced following theadministration of some immunostimulatory nucleic acids, these nucleicacids can be used to generate IFNα endogenously.

[0229] In another embodiment, the asthma/allergy medicament is amedicament selected from the group consisting of PDE-4 inhibitor,bronchodilator/beta-2 agonist, K+ channel opener, VLA-4 antagonist,neurokin antagonist, TXA2 synthesis inhibitor, xanthanine, arachidonicacid antagonist, 5 lipoxygenase inhibitor, thromboxin A2 receptorantagonist, thromboxane A2 antagonist, inhibitor of 5-lipox activationprotein, and protease inhibitor, but is not so limited. In someimportant embodiments, the asthma/allergy medicament is aBronchodilator/beta-2 agonist selected from the group consisting ofsalmeterol, salbutamol, terbutaline, D2522/formoterol, fenoterol, andorciprenaline.

[0230] In another embodiment, the asthma/allergy medicament is amedicament selected from the group consisting of anti-histamines andprostaglandin inducers. In one embodiment, the anti-histamine isselected from the group consisting of loratidine, cetirizine, buclizine,ceterizine analogues, fexofenadine, terfenadine, desloratadine,norastemizole, epinastine, ebastine, ebastine, astemizole,levocabastine, azelastine, tranilast, terfenadine, mizolastine,betatastine, CS 560, and HSR 609. In another embodiment, theprostaglandin inducer is S-5751.

[0231] In yet another embodiment, the asthma/allergy medicament isselected from the group consisting of steroids and immunomodulators. Theimmunomodulators may be selected from the group consisting ofanti-inflammatory agents, leukotriene antagonists, IL-4 muteins, solubleIL-4 receptors, immunosuppressants, anti-IL-4 antibodies, IL-4antagonists, anti-IL-5 antibodies, soluble IL-13 receptor-Fc fusionproteins, anti-IL-9 antibodies, CCR3 antagonists, CCR5 antagonists,VLA-4 inhibitors, and downregulators of IgE, but are not so limited. Inone embodiment, the downregulator of IgE is an anti-IgE. In anotherembodiment, the steroid is selected from the group consisting ofbeclomethasone, fluticasone, tramcinolone, budesonide, and budesonide.In still a further embodiment, the immunosuppressant is a tolerizingpeptide vaccine.

[0232] In one embodiment, the immunostimulatory nucleic acid isadministered concurrently with the asthma/allergy medicament. In anotherembodiment, the subject is an immunocompromised subject.

[0233] Immunostimulatory nucleic acids can be combined with yet othertherapeutic agents such as adjuvants to enhance immune responses. Theimmunostimulatory nucleic acid and other therapeutic agent may beadministered simultaneously or sequentially. When the other therapeuticagents are administered simultaneously they can be administered in thesame or separate formulations, but are administered at the same time.The other therapeutic agents are administered sequentially with oneanother and with immunostimulatory nucleic acid, when the administrationof the other therapeutic agents and the immunostimulatory nucleic acidis temporally separated. The separation in time between theadministration of these compounds may be a matter of minutes or it maybe longer. Other therapeutic agents include but are not limited toadjuvants, cytokines, antibodies, antigens, etc.

[0234] The compositions of the invention may also comprise a non-nucleicacid adjuvants. A non-nucleic acid adjuvant is any molecule or compoundexcept for the immunostimulatory nucleic acids described herein whichcan stimulate the humoral and/or cellular immune response. Non-nucleicacid adjuvants include, for instance, adjuvants that create a depoteffect, immune stimulating adjuvants, and adjuvants that create a depoteffect and stimulate the immune system.

[0235] An adjuvant that creates a depot effect as used herein is anadjuvant that causes the antigen to be slowly released in the body, thusprolonging the exposure of immune cells to the antigen. This class ofadjuvants includes but is not limited to alum (e.g., aluminum hydroxide,aluminum phosphate); or emulsion-based formulations including mineraloil, non-mineral oil, water-in-oil or oil-in-water-in oil emulsion,oil-in-water emulsions such as Seppic ISA series of Montanide adjuvants(e.g., Montanide ISA 720, AirLiquide, Paris, France); MF-59 (asqualene-in-water emulsion stabilized with Span 85 and Tween 80; ChironCorporation, Emeryville, Calif.; and PROVAX (an oil-in-water emulsioncontaining a stabilizing detergent and a micelle-forming agent; IDEC,Pharmaceuticals Corporation, San Diego, Calif.).

[0236] An immune stimulating adjuvant is an adjuvant that causesactivation of a cell of the immune system. It may, for instance, causean immune cell to produce and secrete cytokines. This class of adjuvantsincludes but is not limited to saponins purified from the bark of the Q.saponaria tree, such as QS21 (a glycolipid that elutes in the 21^(st)peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc.,Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer;Virus Research Institute, USA); derivatives of lipopolysaccharides suchas monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc.,Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyldipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related tolipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongationfactor (a purified Leishmania protein; Corixa Corporation, Seattle,Wash.).

[0237] Adjuvants that create a depot effect and stimulate the immunesystem are those compounds which have both of the above- identifiedfunctions. This class of adjuvants includes but is not limited to ISCOMS(immunostimulating complexes which contain mixed saponins, lipids andform virus-sized particles with pores that can hold antigen; CSL,Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2which is an oil-in-water emulsion containing MPL and QS21: SmithKlineBeecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKlineBeecham adjuvant system #4 which contains alum and MPL; SBB, Belgium);non-ionic block copolymers that form micelles such as CRL 1005 (thesecontain a linear chain of hydrophobic polyoxypropylene flanked by chainsof polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex AdjuvantFormulation (SAF, an oil-in-water emulsion containing Tween 80 and anonionic block copolymer; Syntex Chemicals, Inc., Boulder, Colo.).

[0238] The immunostimulatory nucleic acids are themselves useful asadjuvants for inducing a humoral immune response. Thus they can bedelivered to a subject exposed to an antigen to produce an enhancedimmune response to the antigen.

[0239] The immunostimulatory nucleic acids are useful as mucosaladjuvants. It has previously been discovered that both systemic andmucosal immunity are induced by mucosal delivery of CpG nucleic acids.The systemic immunity induced in response to CpG nucleic acids includedboth humoral and cell-mediated responses to specific antigens that werenot capable of inducing systemic immunity when administered alone to themucosa. Furthermore, both CpG nucleic acids and cholera toxin (CT, amucosal adjuvant that induces a Th2-like response) induced CTL. This wassurprising since with systemic immunization, the presence of Th2-likeantibodies is normally associated with a lack of CTL (Schirmbeck et al.,1995). Based on the results presented herein it is expected that theimmunostimulatory nucleic acids will function in a similar manner.

[0240] Additionally, the immunostimulatory nucleic acids induce amucosal response at both local (e.g., lung) and remote (e.g., lowerdigestive tract) mucosal sites. Significant levels of IgA antibodies areinduced at distant mucosal sites by the immunostimulatory nucleic acids.CT is generally considered to be a highly effective mucosal adjuvant. Ashas been previously reported (Snider 1995), CT induces predominantlyIgG1 isotype of antibodies, which are indicative of Th2-type response.In contrast, the immunostimulatory nucleic acids are more Th1 withpredominantly IgG2a antibodies, especially after boost or when the twoadjuvants are combined. Th1-type antibodies in general have betterneutralizing capabilities, and furthermore, a Th2 response in the lungis highly undesirable because it is associated with asthma (Kay, 1996,Hogg, 1997). Thus the use of immunostimulatory nucleic acids as amucosal adjuvant has benefits that other mucosal adjuvants cannotachieve. The immunostimulatory nucleic acids of the invention also areuseful as mucosal adjuvants for induction of both a systemic and amucosal immune response.

[0241] Mucosal adjuvants referred to as non-nucleic acid mucosaladjuvants may also be administered with the immunostimulatory nucleicacids. A non-nucleic acid mucosal adjuvant as used herein is an adjuvantother than a immunostimulatory nucleic acid that is capable of inducinga mucosal immune response in a subject when administered to a mucosalsurface in conjunction with an antigen. Mucosal adjuvants include butare not limited to Bacterial toxins e.g., Cholera toxin (CT), CTderivatives including but not limited to CT B subunit (CTB) (Wu et al.,1998, Tochikubo et al., 1998); CTD53 (Val to Asp) (Fontana et al.,1995); CTK97 (Val to Lys) (Fontana et al., 1995); CTK104 (Tyr to Lys)(Fontana et al., 1995); CTD53/K63 (Val to Asp, Ser to Lys) (Fontana etal., 1995); CTH54 (Arg to His) (Fontana et al., 1995); CTN107 (His toAsn) (Fontana et al., 1995); CTE114 (Ser to Glu) (Fontana et al., 1995);CTE112K (Glu to Lys) (Yamamoto et al., 1997a); CTS61F (Ser to Phe)(Yamamoto et al., 1997a, 1997b); CTS106 (Pro to Lys) (Douce et al.,1997, Fontana et al., 1995); and CTK63 (Ser to Lys) (Douce et al., 1997,Fontana et al., 1995), Zonula occludens toxin, zot, Escherichia coliheat-labile enterotoxin, Labile Toxin (LT), LT derivatives including butnot limited to LT B subunit (LTB) (Verweij et al., 1998); LT7K (Arg toLys) (Komase et al., 1998, Douce et al., 1995); LT61F (Ser to Phe)(Komase et al., 1998); LT112K (Glu to Lys) (Komase et al., 1998); LT118E(Gly to Glu) (Komase et al., 1998); LT146E (Arg to Glu) (Komase et al.,1998); LT192G (Arg to Gly) (Komase et al., 1998); LTK63 (Ser to Lys)(Marchetti et al., 1998, Douce et al., 1997, 1998, Di Tommaso et al.,1996); and LTR72 (Ala to Arg) (Giuliani et al., 1998), Pertussis toxin,PT. (Lycke et al., 1992, Spangler BD, 1992, Freytag and Clemments, 1999,Roberts et al., 1995, Wilson et al., 1995) including PT-9K/129G (Robertset al., 1995, Cropley et al., 1995); Toxin derivatives (see below)(Holmgren et al., 1993, Verweij et al., 1998, Rappuoli et al., 1995,Freytag and Clements, 1999); Lipid A derivatives (e.g., monophosphoryllipid A, MPL) (Sasaki et al., 1998, Vancott et al., 1998; MuramylDipeptide (MDP) derivatives (Fukushima et al., 1996, Ogawa et al., 1989,Michalek et al., 1983, Morisaki et al., 1983); Bacterial outer membraneproteins (e.g., outer surface protein A (OspA) lipoprotein of Borreliaburgdorferi, outer membrane protine of Neisseria meningitidis) (Marinaroet al., 1999, Van de Verg et al., 1996); Oil-in-water emulsions (e.g.,MF59) (Barchfield et al., 1999, Verschoor et al., 1999, O'Hagan, 1998);Aluminum salts (Isaka et al., 1998, 1999); and Saponins (e.g., QS21)Antigenics, Inc., Woburn, Mass.) (Sasaki et al., 1998, MacNeal et al.,1998), ISCOMS, MF-59 (a squalene-in-water emulsion stabilized with Span85 and Tween 80; Chiron Corporation, Emeryville, CA); the Seppic ISAseries of Montanide adjuvants (e.g., Montanide ISA 720; AirLiquide,Paris, France); PROVAX (an oil-in-water emulsion containing astabilizing detergent and a micelle-forming agent; IDEC PharmaceuticalsCorporation, San Diego, Calif.); Syntex Adjuvant Formulation (SAF;Syntex Chemicals, Inc., Boulder, Colo.);poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus ResearchInstitute, USA) and Leishmania elongation factor (Corixa Corporation,Seattle, Wash.).

[0242] Immune responses can also be induced or augmented by theco-administration or co-linear expression of cytokines (Bueler &Mulligan, 1996; Chow et al., 1997; Geissler et al., 1997; Iwasaki etal., 1997; Kim et al., 1997) or B-7 co-stimulatory molecules (Iwasaki etal., 1997; Tsuji et al., 1997) with the immunostimulatory nucleic acids.The cytokines can be administered directly with immunostimulatorynucleic acids or may be administered in the form of a nucleic acidvector that encodes the cytokine, such that the cytokine can beexpressed in vivo. In one embodiment, the cytokine is administered inthe form of a plasmid expression vector. The term cytokine is used as ageneric name for a diverse group of soluble proteins and peptides whichact as humoral regulators at nano- to picomolar concentrations andwhich, either under normal or pathological conditions, modulate thefunctional activities of individual cells and tissues. These proteinsalso mediate interactions between cells directly and regulate processestaking place in the extracellular environment.

[0243] Examples of cytokines include, but are not limited to IL-1, IL-2,IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18,granulocyte-macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), interferon-γ (γ-IFN), IFN-α, tumornecrosis factor (TNF), TGF-β, FLT-3 ligand, and CD40 ligand.

[0244] Cytokines play a role in directing the T cell response. Helper(CD4+) T cells orchestrate the immune response of mammals throughproduction of soluble factors that act on other immune system cells,including other T cells. Most mature CD4+T helper cells express one oftwo cytokine profiles: Th1 or Th2. The Th1 subset promotes delayed-typehypersensitivity, cell-mediated immunity, and immunoglobulin classswitching to IgG_(2a). The Th2 subset induces humoral immunity byactivating B cells, promoting antibody production, and inducing classswitching to IgG₁ and IgE. In some embodiments, it is preferred that thecytokine be a Th1 cytokine.

[0245] The immunostimulatory nucleic acids may be directly administeredto the subject or may be administered in conjunction with a nucleic aciddelivery complex. A nucleic acid delivery complex shall mean a nucleicacid molecule associated with (e.g. ionically or covalently bound to; orencapsulated within) a targeting means (e.g. a molecule that results inhigher affinity binding to target cell (e.g., B cell surfaces and/orincreased cellular uptake by target cells). Examples of nucleic aciddelivery complexes include nucleic acids associated with a sterol (e.g.cholesterol), a lipid (e.g. a cationic lipid, virosome or liposome), ora target cell specific binding agent (e.g. a ligand recognized by targetcell specific receptor). Preferred complexes may be sufficiently stablein vivo to prevent significant uncoupling prior to internalization bythe target cell. However, the complex can be cleavable under appropriateconditions within the cell so that the nucleic acid is released in afunctional form.

[0246] Delivery vehicles or delivery devices for delivering antigen andnucleic acids to surfaces have been described. The Immunostimulatorynucleic acid and/or the antigen and/or other therapeutics may beadministered alone (e.g., in saline or buffer) or using any deliveryvehicles known in the art. For instance the following delivery vehicleshave been described: Cochleates (Gould-Fogerite et al., 1994, 1996);Emulsomes (Vancott et al., 1998, Lowell et al., 1997); ISCOMs (Mowat etal., 1993, Carlsson et al., 1991, Hu et., 1998, Morein et al., 1999);Liposomes (Childers et al., 1999, Michalek et al., 1989, 1992, de Haan1995a, 1995b); Live bacterial vectors (e.g., Salmonella, Escherichiacoli, Bacillus calmatte-guerin, Shigella, Lactobacillus) (Hone et al.,1996, Pouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991,Nugent et al., 1998); Live viral vectors (e.g., Vaccinia, adenovirus,Herpes Simplex) (Gallichan et al., 1993, 1995, Moss et al., 1996, Nugentet al., 1998, Flexner et al., 1988, Morrow et al., 1999); Microspheres(Gupta et al., 1998, Jones et al., 1996, Maloy et al., 1994, Moore etal., 1995, O'Hagan et al., 1994, Eldridge et al., 1989); Nucleic acidvaccines (Fynan et al., 1993, Kukin et al., 1997, Sasaki et al., 1998,Okada et al., 1997, Ishii et al., 1997); Polymers (e.g.carboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill etal., 1998); Polymer rings (Wyatt et al., 1998); Proteosomes (Vancott etal., 1998, Lowell et al., 1988, 1996, 1997); Sodium Fluoride (Hashi etal., 1998); Transgenic plants (Tacket et al., 1998, Mason et al., 1998,Haq et al., 1995); Virosomes (Gluck et al., 1992, Mengiardi et al.,1995, Cryz et al., 1998); Virus-like particles (Jiang et al., 1999,Leibl et al., 1998). Other delivery vehicles are known in the art andsome additional examples are provided below in the discussion ofvectors.

[0247] The stimulation index of a particular immunostimulatory nucleicacid can be tested in various immune cell assays. Preferably, thestimulation index of the immunostimulatory nucleic acid with regard to Bcell proliferation is at least about 5, preferably at least about 10,more preferably at least about 15 and most preferably at least about 20as determined by incorporation of ³H uridine in a murine B cell culture,which has been contacted with 20 μM of nucleic acid for 20 h at 37° C.and has been pulsed with 1 μCi of ³H uridine; and harvested and counted4 h later as described in detail in PCT Published Patent ApplicationsPCT/US95/01570 (WO 96/02555) and PCT/US97/19791 (WO 98/18810) claimingpriority to U.S. Serial Nos. 08/386,063 and 08/960,774, filed on Feb. 7,1995 and Oct. 30, 1997 respectively. For use in vivo, for example, it isimportant that the immunostimulatory nucleic acids be capable ofeffectively inducing an immune response, such as, for example, antibodyproduction. Other assays designed to test efficacy and effective amountsare described in the Examples.

[0248] Immunostimulatory nucleic acids are effective in non-rodentvertebrate. Different immunostimulatory nucleic acid can cause optimalimmune stimulation depending on the type of subject and the sequence ofthe immunostimulatory nucleic acid. Many vertebrates have been foundaccording to the invention to be responsive to the same class ofimmunostimulatory nucleic acids, sometimes referred to as human specificimmunostimulatory nucleic acids. Rodents, however, respond to differentnucleic acids.

[0249] As shown herein an immunostimulatory nucleic acid causing optimalstimulation in humans may not generally cause optimal stimulation in amouse and vice versa. An immunostimulatory nucleic acid causing optimalstimulation in humans often does, however, cause optimal stimulation inother animals such as cow, horses, sheep, etc. One of skill in the artcan identify the optimal nucleic acid sequences useful for a particularspecies of interest using routine assays described herein and/or knownin the art, using the guidance supplied herein.

[0250] The term effective amount of a immunostimulatory nucleic acidrefers to the amount necessary or sufficient to realize a desiredbiologic effect. For example, an effective amount of a immunostimulatorynucleic acid for inducing mucosal immunity is that amount necessary tocause the development of IgA in response to an antigen upon exposure tothe antigen, whereas that amount required for inducing systemic immunityis that amount necessary to cause the development of IgG in response toan antigen upon exposure to the antigen. Combined with the teachingsprovided herein, by choosing among the various active compounds andweighing factors such as potency, relative bioavailability, patient bodyweight, severity of adverse side-effects and preferred mode ofadministration, an effective prophylactic or therapeutic treatmentregimen can be planned which does not cause substantial toxicity and yetis entirely effective to treat the particular subject. The effectiveamount for any particular application can vary depending on such factorsas the disease or condition being treated, the particularimmunostimulatory nucleic acid being administered, the antigen, the sizeof the subject, or the severity of the disease or condition. One ofordinary skill in the art can empirically determine the effective amountof a particular immunostimulatory nucleic acid and/or antigen and/orother therapeutic agent without necessitating undue experimentation.

[0251] Subject doses of the compounds described herein for mucosal orlocal delivery typically range from about 0.1 μg to 10 mg peradministration, which depending on the application could be given daily,weekly, or monthly and any other amount of time therebetween. Moretypically mucosal or local doses range from about 10 μg to 5 mg peradministration, and most typically from about 100 μg to 1 mg, with 2-4administrations being spaced days or weeks apart. More typically, immunestimulant doses range from 1 μg to 10 mg per administration, and mosttypically 10 μg to 1 mg, with daily or weekly administrations. Subjectdoses of the compounds described herein for parenteral delivery for thepurpose of inducing an antigen-specific immune response, wherein thecompounds are delivered with an antigen but not another therapeuticagent are typically 5 to 10,000 times higher than the effective mucosaldose for vaccine adjuvant or immune stimulant applications, and moretypically 10 to 1,000 times higher, and most typically 20 to 100 timeshigher. Doses of the compounds described herein for parenteral deliveryfor the purpose of inducing an innate immune response or for increasingADCC or for inducing an antigen specific immune response when theimmunostimulatory nucleic acids are administered in combination withother therapeutic agents or in specialized delivery vehicles typicallyrange from about 0.1 μg to 10 mg per administration, which depending onthe application could be given daily, weekly, or monthly and any otheramount of time therebetween. More typically parenteral doses for thesepurposes range from about 10 μg to 5 mg per administration, and mosttypically from about 100 μg to 1 mg, with 2-4 administrations beingspaced days or weeks apart. In some embodiments, however, parenteraldoses for these purposes may be used in a range of 5 to 10,000 timeshigher than the typical doses described above.

[0252] For any compound described herein the therapeutically effectiveamount can be initially determined from animal models. A therapeuticallyeffective dose can also be determined from human data for CpGoligonucleotides which have been tested in humans (human clinical trialshave been initiated) and for compounds which are known to exhibitsimilar pharmacological activities, such as other mucosal adjuvants,e.g., LT and other antigens for vaccination purposes, for the mucosal orlocal administration. Higher doses are required for parenteraladministration. The applied dose can be adjusted based on the relativebioavailability and potency of the administered compound. Adjusting thedose to achieve maximal efficacy based on the methods described aboveand other methods as are well-known in the art is well within thecapabilities of the ordinarily skilled artisan.

[0253] The formulations of the invention are administered inpharmaceutically acceptable solutions, which may routinely containpharmaceutically acceptable concentrations of salt, buffering agents,preservatives, compatible carriers, adjuvants, and optionally othertherapeutic ingredients.

[0254] For use in therapy, an effective amount of the immunostimulatorynucleic acid can be administered to a subject by any mode that deliversthe nucleic acid to the desired surface, e.g., mucosal, systemic.Administering the pharmaceutical composition of the present inventionmay be accomplished by any means known to the skilled artisan. Preferredroutes of administration include but are not limited to oral,parenteral, intramuscular, intranasal, intratracheal, inhalation,ocular, vaginal, and rectal.

[0255] For oral administration, the compounds (i.e., immunostimulatorynucleic acids, antigens and other therapeutic agents) can be formulatedreadily by combining the active compound(s) with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable thecompounds of the invention to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a subject to be treated. Pharmaceutical preparationsfor oral use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

[0256] Dragee cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

[0257] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art.

[0258] All formulations for oral administration should be in dosagessuitable for such administration.

[0259] For buccal administration, the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0260] For administration by inhalation, the compounds for use accordingto the present invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0261] The compounds, when it is desirable to deliver them systemically,may be formulated for parenteral administration by injection, e.g., bybolus injection or continuous infusion. Formulations for injection maybe presented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

[0262] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

[0263] Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0264] The compounds may also be formulated in rectal or vaginalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

[0265] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0266] The pharmaceutical compositions also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

[0267] Suitable liquid or solid pharmaceutical preparation forms are,for example, aqueous or saline solutions for inhalation,microencapsulated, encochleated, coated onto microscopic gold particles,contained in liposomes, nebulized, aerosols, pellets for implantationinto the skin, or dried onto a sharp object to be scratched into theskin. The pharmaceutical compositions also include granules, powders,tablets, coated tablets, (micro)capsules, suppositories, syrups,emulsions, suspensions, creams, drops or preparations with protractedrelease of active compounds, in whose preparation excipients andadditives and/or auxiliaries such as disintegrants, binders, coatingagents, swelling agents, lubricants, flavorings, sweeteners orsolubilizers are customarily used as described above. The pharmaceuticalcompositions are suitable for use in a variety of drug delivery systems.For a brief review of methods for drug delivery, see Langer, Science249:1527-1533, 1990, which is incorporated herein by reference.

[0268] The immunostimulatory nucleic acids and optionally othertherapeutics and/or antigens may be administered per se (neat) or in theform of a pharmaceutically acceptable salt. When used in medicine thesalts should be pharmaceutically acceptable, but non-pharmaceuticallyacceptable salts may conveniently be used to prepare pharmaceuticallyacceptable salts thereof. Such salts include, but are not limited to,those prepared from the following acids: hydrochloric, hydrobromic,sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulphonic, tartaric, citric, methane sulphonic, formic, malonic,succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, suchsalts can be prepared as alkaline metal or alkaline earth salts, such assodium, potassium or calcium salts of the carboxylic acid group.

[0269] Suitable buffering agents include: acetic acid and a salt (1-2%w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5%w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitablepreservatives include benzalkonium chloride (0.003-0.03% w/v);chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal(0.004-0.02% w/v).

[0270] As described in greater detail herein, the pharmaceuticalcompositions of the invention contain an effective amount of aimmunostimulatory nucleic acid and optionally antigens and/or othertherapeutic agents optionally included in a pharmaceutically-acceptablecarrier. The term pharmaceutically-acceptable carrier means one or morecompatible solid or liquid filler, diluents or encapsulating substanceswhich are suitable for administration to a human or other vertebrateanimal. The term carrier denotes an organic or inorganic ingredient,natural or synthetic, with which the active ingredient is combined tofacilitate the application. The components of the pharmaceuticalcompositions also are capable of being commingled with the compounds ofthe present invention, and with each other, in a manner such that thereis no interaction which would substantially impair the desiredpharmaceutical efficiency.

[0271] The immunostimulatory nucleic acids useful in the invention maybe delivered in mixtures with additional adjuvant(s), othertherapeutics, or antigen(s). A mixture may consist of several adjuvantsin addition to the immunostimulatory nucleic acid or several antigens orother therapeutics.

[0272] A variety of administration routes are available. The particularmode selected will depend, of course, upon the particular adjuvants orantigen selected, the particular condition being treated and the dosagerequired for therapeutic efficacy. The methods of this invention,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of an immune response without causing clinically unacceptableadverse effects. Preferred modes of administration are discussed above.

[0273] The compositions may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing the compounds intoassociation with a carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the compounds into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product. Liquid dose units are vials or ampoules.Solid dose units are tablets, capsules and suppositories. For treatmentof a patient, depending on activity of the compound, manner ofadministration, purpose of the immunization (i.e., prophylactic ortherapeutic), nature and severity of the disorder, age and body weightof the patient, different doses may be necessary. The administration ofa given dose can be carried out both by single administration in theform of an individual dose unit or else several smaller dose units.Multiple administration of doses at specific intervals of weeks ormonths apart is usual for boosting the antigen-specific responses.

[0274] Other delivery systems can include time-release, delayed releaseor sustained release delivery systems. Such systems can avoid repeatedadministrations of the compounds, increasing convenience to the subjectand the physician. Many types of release delivery systems are availableand known to those of ordinary skill in the art. They include polymerbase systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides.

[0275] Microcapsules of the foregoing polymers containing drugs aredescribed in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are: lipids including sterols suchas cholesterol, cholesterol esters and fatty acids or neutral fats suchas mono-di-and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which an agent of the invention is contained in a form withina matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189,and 5,736,152, and (b) diff-usional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-basedhardware delivery systems can be used, some of which are adapted forimplantation.

[0276] The present invention is further illustrated by the followingExamples, which in no way should be construed as further limiting. Theentire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference.

EXAMPLES

[0277] Summary:

[0278] This report summarizes in vitro data with human cellsdemonstrating that ODN 10105 behaves as well or better than ODN 7909 inhuman cell assays. In addition, ODN 10105 behaves as well or better thanODN 7909 in in vitro and in vivo data in mice demonstrating that CpG ODN10105 is useful in the activation of the innate immune system, and inaugmenting humoral and cellular HBsAg-specific responses in mice whencoadministered with the antigen.

[0279] The assays performed were receptor engagement (TLR9), B cellactivation (expression of cell surface activation marker and B cellproliferation) and cytokine secretion (IL-10, IP-10 and IFN-α). Allassays demonstrated that ODN 10105 has properties that were similar orsuperior to ODN 7909. In vitro studies (i.e. B cell proliferationassays, NK lytic activity, cytokine secretion profiles) were carried outusing naive BALB/c mouse splenocytes. In vivo comparison studies werecarried out by comparing the potential of these two ODNs to enhanceantigen specific immune responses to hepatitis B antigen (HBsAg).

[0280] Materials and Methods:

[0281] Human Studies

[0282] Oligodeoxynucleotides:

[0283] All ODNs (10105, 7909 and control ODN) were provided by ColeyPharmaceutical GmbH (Langenfeld, Germany). The control ODN contained nostimulatory CpG motif. ODNs were diluted in phosphate-buffered saline,and stored at −20° C. All dilutions were carried out using pyrogen-freereagents.

[0284] TLR9 assay:

[0285] Cells used for this assay were expressing the human TLR9 receptorand containing a reporter gene construct. Cells were incubated with ODNsfor 16 h. Each data point was done in triplicate. Cells were lysed andassayed for reporter gene activity. Stimulation indices were calculatedin reference to reporter gene activity of medium without addition ofODN.

[0286] Cell purification:

[0287] Peripheral blood buffy coat preparations from healthy humandonors were obtained from the German Red Cross (Rathingen, Germany) andfrom these, PBMC were purified by centrifugation over Ficoll-Hypaque(Sigma, Germany). The purified PBMC were either used fresh or weresuspended in freezing medium and stored at −70° C. When required,aliquots of these cells were thawed, washed and resuspended in RPMI 1640culture medium supplemented with 10% (v/v) heat inactivated FCS, 1.5 mML-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.

[0288] Cytokine detection:

[0289] Thawed or fresh PBMC were resuspended at a concentration of5×10⁶/ml and added to 48 well flat-bottomed plates (1 ml/well), whichhad previously received nothing or ODN in a variety of concentrations.The cells were cultured in a humidified incubator at 37° C. Culturesupernatants were collected after the indicated time points. If not usedimmediately, supernatants were frozen at −20° C. until required.

[0290] Amounts of cytokines in the supernatants were assessed usingcommercially available ELISA Kits (IL-10; Diaclone, USA) or in-houseELISAs (IP-10 and IFN-α) developed using commercially availableantibodies (from Pharmingen or PBL; Germany or USA, respectively).

[0291] Cultures for flow cytometric analysis of B cell activation:

[0292] Monoclonal antibodies to CD19 and CD86 were purchased from BectonDickinson (Germany). PBMC were incubated for 48 hours with or withoutthe addition of different concentrations of ODNs. B cells wereidentified by expression of CD19 by flow cytometry. Flow cytometric datawere acquired on a FACSCalibur (Becton Dickinson). Data were analyzedusing the computer program CellQuest (Becton Dickinson). ProliferatingCD19 positive B cells were identified after culturing CFSE-labelled PBMC(CFSE is a fluorescing dye binding to all cell surfaces) by decreasedCFSE content using flow cytometry methodology (see above).

[0293] Murine In Vitro/In Vivo Studies:

[0294] Oligodeoxynucleotides:

[0295] CpG ODN 7909 (GMP quality) and 10105 were supplied by ColeyPharmaceutical Inc. (Wellesley, Mass.). All ODN were re suspended insterile, endotoxin free TE at pH 8.0 (OmniPer®; EM S cience, Gibbstown,N.J.) and stored and handle under aseptic conditions to prevent bothmicrobial and endotoxin contamination.

[0296] Dilution of ODNs for assays was carried out in sterile, endotoxinfree PBS, pH 7.2 (Sigma Chemical Company, St. Lois, Mo.).

[0297] Animals:

[0298] Female BALB/c mice (6-8 weeks of age) were used for allexperiments. Animals were purchased from Charles River Canada (Quebec,Canada) and housed in micro isolators at the animal care facility of theOttawa Hospital Research Institute, Civic Site.

[0299] In vitro assays:

[0300] Naïve BALB/c mouse splenocytes were used for all in vitro assays.Animals were anesthetized with isofluorane and euthanized by cervicaldislocation. Spleens were removed under aseptic conditions and placed inPBS+0.2% bovine serum albumin (Sigma Chemical Company). Spleens werethen homogenized and splenocytes were re-suspended in RPMI 1640 (LifeTechnologies, Grand Island, N.Y.) tissue culture medium supplementedwith 2% normal mouse serum (Cedarlane Laboratories, Ontario, Canada),penicillin-streptomycin solution (final concentration of 1000 U/ml and 1mg/ml respectively; Sigma Chemical Company), and 5×10⁻⁵ Mβ-mercaptoethanol (Sigma Chemical Company).

[0301] B cell proliferation assays:

[0302] Spleen cell suspensions were prepared and adjusted to a finalconcentration of 5×10⁶ cells per ml in complete RPMI 1640. Splenocytesuspension was plated onto 96-well U-bottom tissue culture plates (100μl/well) along with 100 μl of each stimulant diluted to appropriateconcentrations in complete RPMI 1640. The stimulants used were CpG ODN(at 1, 3, 10 μg/ml) 7909 and 10105. Concanavalin A (10 μg/ml, SigmaChemical Company) LPS (10 μg/ml, Sigma Chemical Company) were used aspositive controls and cells cultured with media alone were used asnegative controls. Each splenocyte sample was plated in triplicate andcells were incubated in a humidified 5% CO₂ incubator at 37° C. for 96hr. At the end of the incubation period, cells were pulsed with³H-thymidine (20 μCi/ml) at 96 hr post incubation for 16 hours,harvested and measured for radioactivity.

[0303] Cytokine secretion profiles:

[0304] Spleen cell suspensions were prepared and plated in 96-wellU-bottom tissue culture plates as described for B cell proliferationassays. Each splenocyte sample was plated in triplicate and the cellswere incubated in a humidified 5% CO₂ incubator at 37° C. for 6, 12 or48 hr. At the end of the incubation period, 96-well plates werecentrifuged for 5 min at 1200 rpm and culture supernatants harvested andstored at −80° C. until assayed. Commercially available assay kits(mouse OptEIA kits; PharMingen, Mississauga, ON) were used according tomanufacturers instructions to assay cytokine levels in culturesupernatants taken at 6 hr (TNF-α), 24 hr (IL-12) and 48 hr (IL-6 andIL-10).

[0305] NK assays:

[0306] Splenocyte suspensions were prepared as described previously andadjusted to a final concentration of 3×10⁶ cells per ml in complete RPMI1640. Splenocyte suspension (10 ml; 30×10⁶ cells) was plated in T-25tissue culture flasks (Fisher Scientific, Ottawa, ON) along with eitherCpG ODN (at 1, 3, 10 μg/ml) 7909 and 10105. Splenocytes cultured withmedia alone was used as negative controls. Each splenocyte culture wasincubated in a humidified 5% CO₂ incubator at 37° C. for 24 hr. At theend of the incubation period, cells were plated at differenteffector:target ratios onto 96-well U-bottom tissue culture plates (100μl/well) along with 100 μl of ⁵¹Cr labeled target cells at 5×10⁴cells/ml. NK sensitive mouse lymphoma cell line YAC-1 (ATCC # TIB-160,ATCC, Manassas, Va.) was used as the target cell line. Each sample wasplated in triplicate and the cells were incubated in a humidified 5% CO₂incubator at 37° C. for 4 hr. Target cells were incubated with mediaalone or with 2N HCl to determine spontaneous release and maximumrelease respectively. At the end of the incubation period, supernatantswere harvested and radioactivity levels were determined using a γcounter. The % lysis was determined using the following formula;${\% \quad {specific}\quad {release}} = {\frac{{{experimental}\quad {release}} - {{spontaneous}\quad {release}}}{{{maximum}\quad {release}} - {{spontaneous}\quad {release}}} \times 100}$

[0307] Immunization of mice:

[0308] BALB/c mice (n=10/group) were immunized with 1 μg HBsAg sub typead (International Enzymes, CA) alone or in combination with either 10 μgCpG ODN 7909 or CpG ODN 10105. Animals were bled and boosted at 4 weekspost-primary immunization. At 1 week post boost 5 animals from eachgroup was euthanized and spleens removed for CTL assays

[0309] Determination of antibody responses:

[0310] Antibodies (total IgG, IgG1 and IgG2a) specific to HBsAg(anti-HBs) were detected and quantified by endpoint dilution ELISAassay, which was performed in triplicate on samples from individualanimals. End-point titers were defined as the highest plasma dilutionthat resulted in an absorbance value (OD 450) two times greater thanthat of non-immune plasma with a cut-off value of 0.05. These werereported as group mean titers ±SEM.

[0311] Statistical analysis:

[0312] Statistical analysis was performed using InStat program (GraphPAD Software, San Diego). The statistical difference between groups weredetermined by Student's t test (for two groups) or by 1-factor ANOVAfollowed by Tukey's test (for three or more groups) on raw data ortransformed data (logio, for heteroscedastic populations).

[0313] Results:

[0314] TLR9 engagement:

[0315] Recently the receptor for the recognition of CpG sequences wasidentified and shown to be a member of the Toll-Like Receptor (TLR)family (Hemmi et al., 2000). This receptor, TLR9, is readily activatedby ODNs containing optimal immunostimulatory CpG sequences. We incubateda cell line stably expressing the human TLR9 with differentconcentrations of ODNs 7909 and 10105 as well as a control ODN (FIG. 1).

[0316] The results demonstrate that there was no statisticallysignificant difference between the two B class ODNs in activating TLR9.Both ODNs showed the same dose-response curve and reached maximumactivation at the same concentrations. The control ODN used did notinduce TLR9 activation even at the highest concentration of 24 μg/ml.

[0317] Human B cells:

[0318] One characteristic of type B ODNs is their ability to veryefficiently activate B cells (Krieg et al., 1995). B cells andplasmacytoid DC are at the moment the only immune cell types known toexpress TLR9 (Krug et al., 2001; Bauer et al., 2001). The directactivation of B cells induced by ODNs 7909 and 10105 was measured by upregulation of the cell surface marker CD86 (FIG. 2), and proliferationof B cells (FIG. 3). For CD86 expression on human B cells PBMC ofhealthy blood donors were incubated with different ODNs and B cellactivation measured as described in Materials and Methods.

[0319] The results demonstrate that 10105 as well as 7909 are verypotent stimulators of human B cells. FIG. 2 shows that these CpG ODNswere capable to stimulate B cells at an in vitro concentration of only0.4 μg/ml. The plateau was reached at about 1.6 μg/ml and more than 70%of B cells were found to have up regulated CD86 in contrast to thecontrol that was much less potent. A similar result was obtained for theinduction of B cell proliferation, with exception that 10105 was able toinduce B cell proliferation even at the highest dose test of 6 μg/ml,while 7909 plateaued at the dose of 1.6 to 3.0 μg/ml (FIG. 3).

[0320] Cytokine secretion:

[0321] ODNs of the B class induce a Th1 dominated immune response invivo as well as in vitro. It was found that they are able to inducetypical Th1 cytokines such as IFN-γ and IFN-α as well as chemokines suchas MCP-1 and IP-10. In addition, low secretion of the pro-inflammatorycytokines IL-6 as well as TNF-α and secretion of the negative regulatorIL-10 can be observed. The secretion of the Th1 cytokine IFN-α, thechemokine IP-10 as well as the regulatory cytokine IL-10 and thepro-inflammatory cytokine TNF-α were measured following administrationof 10105 and 7909. FIG. 4 shows the result for an experiment performedwith 6 different donors at 0.2, 0.4 and 1.6 μg/ml to measure in vitroIFN-α secretion.

[0322] Both CpG ODNs induced high levels of IFN-α with a maximum reachedat 0.4 to 1.6μg/ml. In contrast, the control ODN induced low amounts ofIFN-α starting only at 5.0 μg/ml. ODN 10105 induced higher levels ofIFN-α at both the 1.6 and 5.0 μg/ml doses, as compared to ODN 7909. TheODNs 7909 and 10105, in contrast to the control ODN, induced thechemokine IP-10 as shown in FIG. 5, again with ODN 10105 inducing higherlevels at the 0.4 μg/ml dose.

[0323] The time-dependent effects on different cytokines were alsoanalyzed. Therefore, PBMC from different donors were incubated for 8 h,24 h, 36 h and/or 48 h and the secretion of IL-10 or IFN-α was measured.FIGS. 6 and 7 demonstrate the results obtained for IFN-α with twodifferent donors upon incubation for 8 h and 24 h (FIG. 6) or 36 h and48 h (FIG. 7). IFN-α was initially secreted as early as 8 h uponincubation with CpG ODN, maximum amounts were reached at 24 h and theamounts stayed at that level or even increased between 24 h and 48 h.LPS did not induce any IFN-α. For both donors, the ODN 10105 stimulatedhigher levels of FN-α at the 8 hour time point at a concentration of 1.6μg/ml.

[0324] A very similar experiment was performed for IL-10 secretion(FIGS. 8 and 9). This cytokine showed similar characteristics to IFN-αalthough maximum amounts were obtained at a 48 h. Again, as demonstratedabove for IFN-α, both CpG ODNs 7909 and 10105 demonstrated almostidentical properties in these as in all other assays performed.

[0325] In vitro mouse studies:

[0326] As shown in FIG. 10, ODN 10105 is able to stimulate higher levelsof B cell proliferation than ODN 7909 at all concentrations tested.According to the data shown in FIG. 11, both CpG ODN 7909 and 10105 areable to stimulate IL-10, IL-12, IL-6 and TNF-secretion. For IL-12 andTNF-secretion, ODN 10105 elicits more factor secretion at allconcentrations tested than does 7909.

[0327] The CpG ODN have essentially equal potency in enhancing lyticactivity of NK cells in mouse splenocyte cultures (FIG. 12).

[0328] As shown in FIG. 13, either CpG ODN 7909 or 10105 significantlyenhanced antibody titers against HBsAg compared to antigen alone(p<0.0001) whereas there was no significant increase in anti-HBsresponses when control ODN was used in combination with HBsAg (p=0.85).

[0329] As shown in FIG. 14, the increase in total IgG levels is similarboth the CpG ODN.

[0330] In mice IgG isotype distribution is widely used as an indicationof the nature of the immune response where high IgG2a/IgG1 ratios areindicative of a Th1 biased immune response (Constant and Bottomly,1997). In the present study, the use of CpG ODN significantly enhancedIgG2a titers compared to when antigen was used alone or in combinationwith control ODN 2137 (p<0.001 for Ag vs. 7909 or 101 05 and p<0.01 forAg+7909 vs. Ag+2137 and p<0.05 for Ag+10105 vs. Ag+2137). However, thelevel of IgG2a response was similar when either CpG ODN 7909 or 10105was used in combination with HBsAg (p>0.05). Therefore, both CpG ODN7909 and 10105 are equally potent in their ability to induce Th1 biasedimmune responses as measured by the increased levels of IgG2a over IgG1.

[0331] Conclusions:

[0332] In vitro data with human cells demonstrating that ODN 10105behaves similarly and in some instances, in a manner superior to that ofODN 7909 is demonstrated. According to the results of the murinestudies, CpG ODN 7909 and 10105 have similar immune potentiatingproperties, both for their in vitro effects on innate immune responsesas well as their ability to augment antigen specific responses in vivowhen administered together with an antigen.

[0333] References

[0334] 1. Bauer, S. et al.; Human TLR9 confers responsiveness tobacterial DNA via species-specific CpG motif recognition; PNAS 98, 2001.

[0335] 2. Constant, S. L., and K. Bottomly 1997. Induction of Th1 andTh2 CD4+T cell responses: the alternative approaches Annu Rev Immunol.15:297-322.

[0336] 3. Hemmi, H. et al.; A Toll-like receptor recognizes bacterialDNA; Nature 408, 2000.

[0337] 4. Krieg, A. M. et al.; CpG motifs in bacterial DNA triggerdirect B-cell activation; Nature 374, 1995.

[0338] 5. Krug, A. et al.; Toll-like receptor expression reveals CpG DNAas a unique microbial stimulus for pDC which synergizes with CD40 ligandto induce high amounts of IL-12; Eur. J. Immunol. 31; 2001.

Equivalents

[0339] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by examplesprovided, since the examples are intended as a single illustration ofone aspect of the invention and other functionally equivalentembodiments are within the scope of the invention. Various modificationsof the invention in addition to those shown and described herein willbecome apparent to those skilled in the art from the foregoingdescription and fall within the scope of the appended claims. Theadvantages and objects of the invention are not necessarily encompassedby each embodiment of the invention.

1 24 1 24 DNA Artificial sequence Oligodeoxynucleotide 1 tcgtcgttttgtcgtttttt tcga 24 2 24 DNA Artificial sequence Oligodeoxynucleotide 2tcgtcgtttt gtcgttttgt cgtt 24 3 24 DNA Artificial sequenceOligodeoxynucleotide 3 nnnnnnnnnn nnnnnttttt tcga 24 4 9 DNA Artificialsequence Oligodeoxynucleotide 4 ttttttcga 9 5 24 DNA Artificial sequenceOligodeoxynucleotide 5 tcgtcgtttt gtcgtttttn nnnn 24 6 19 DNA Artificialsequence Oligodeoxynucleotide 6 tcgtcgtttt gtcgttttt 19 7 23 DNAArtificial sequence Oligodeoxynucleotide 7 tcgtcgtttt gtcgtttttt tcg 238 22 DNA Artificial sequence Oligodeoxynucleotide 8 tcgtcgttttgtcgtttttt tc 22 9 21 DNA Artificial sequence Oligodeoxynucleotide 9tcgtcgtttt gtcgtttttt t 21 10 20 DNA Artificial sequenceOligodeoxynucleotide 10 tcgtcgtttt gtcgtttttt 20 11 23 DNA Artificialsequence Oligodeoxynucleotide 11 cgtcgttttg tcgttttttt cga 23 12 22 DNAArtificial sequence Oligodeoxynucleotide 12 gtcgttttgt cgtttttttc ga 2213 21 DNA Artificial sequence Oligodeoxynucleotide 13 tcgttttgtcgtttttttcg a 21 14 20 DNA Artificial sequence Oligodeoxynucleotide 14cgttttgtcg tttttttcga 20 15 19 DNA Artificial sequenceOligodeoxynucleotide 15 gttttgtcgt ttttttcga 19 16 18 DNA Artificialsequence Oligodeoxynucleotide 16 ttttgtcgtt tttttcga 18 17 17 DNAArtificial sequence Oligodeoxynucleotide 17 tttgtcgttt ttttcga 17 18 16DNA Artificial sequence Oligodeoxynucleotide 18 ttgtcgtttt tttcga 16 1915 DNA Artificial sequence Oligodeoxynucleotide 19 tgtcgttttt ttcga 1520 14 DNA Artificial sequence Oligodeoxynucleotide 20 gtcgtttttt tcga 1421 13 DNA Artificial sequence Oligodeoxynucleotide 21 tcgttttttt cga 1322 12 DNA Artificial sequence Oligodeoxynucleotide 22 cgtttttttc ga 1223 11 DNA Artificial sequence Oligodeoxynucleotide 23 gtttttttcg a 11 2410 DNA Artificial sequence Oligodeoxynucleotide 24 tttttttcga 10

We claim:
 1. A composition comprising an immunostimulatory nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1.
 2. Thecomposition of claim 1, wherein the immunostimulatory nucleic acidmolecule consists of the nucleotide sequence of SEQ ID NO:1.
 3. Thecomposition of claim 1, further comprising an antigen.
 4. Thecomposition of claim 3, wherein the antigen is selected from the groupconsisting of a microbial antigen, a cancer antigen, and an allergen. 5.The composition of claim 4, wherein the microbial antigen is selectedfrom the group consisting of a bacterial antigen, a viral antigen, afungal antigen and a parasitic antigen.
 6. The composition of claim 3,wherein the antigen is encoded by a nucleic acid vector.
 7. Thecomposition of claim 3, wherein the nucleic acid vector is separate fromthe immunostimulatory nucleic acid.
 8. The composition of claim 3,wherein the antigen is a peptide antigen.
 9. The composition of claim 1,further comprising an adjuvant.
 10. The composition of claim 9, whereinthe adjuvant is a mucosal adjuvant.
 11. The composition of claim 1,further comprising a cytokine.
 12. The composition of claim 1, furthercomprising a therapeutic agent selected from the group consisting of ananti-microbial agent, an anti-cancer agent, an allergy/asthmamedicament.
 13. The composition of claim 12, wherein the anti-microbialagent is selected from the group consisting of an anti-bacterial agent,an anti-viral agent, an anti-fungal agent, and an anti-parasite agent.14. The composition of claim 12, wherein the anti-cancer agent isselected from the group consisting of a chemotherapeutic agent, a cancervaccine, and an immunotherapeutic agent.
 15. The composition of claim12, wherein the allergy/asthma medicament is selected from the groupconsisting of PDE-4 inhibitor, bronchodilator/beta-2 agonist, K+channelopener, VLA-4 antagonist, neurokin antagonist, TXA2 synthesis inhibitor,xanthanine, arachidonic acid antagonist, 5 lipoxygenase inhibitor,thromboxin A2 receptor antagonist, thromboxane A2 antagonist, inhibitorof 5-lipox activation protein, and protease inhibitor.
 17. Thecomposition of claim 1, wherein the immunostimulatory nucleic acid has anucleotide backbone which includes at least one backbone modification.18. The composition of claim 17, wherein the backbone modification is aphosphorothioate modification.
 19. The composition of claim 17, whereinthe nucleotide backbone is chimeric.
 20. The composition of claim 17,wherein the nucleotide backbone is entirely modified.
 21. Thecomposition of claim 1, further comprising a pharmaceutically acceptablecarrier.
 22. The composition of claim 1, wherein the immunostimulatorynucleic acid is free of methylated CpG dinucleotides.
 23. Thecomposition of claim 1, wherein the immunostimulatory nucleic acidincludes at least four CpG motifs.
 24. The composition of claim 1,wherein the immunostimulatory nucleic acid is T-rich.
 25. Thecomposition of claim 1, wherein the immunostimulatory nucleic acidincludes a poly-T sequence.
 26. The composition of claim 1, wherein theimmunostimulatory nucleic acid includes a poly-G sequence.
 27. Thecomposition of claim 1, wherein the immunostimulatory nucleic acid isformulated for oral administration.
 28. The composition of claim 1,wherein the immunostimulatory nucleic acid is formulated as anutritional supplement.
 29. The composition of claim 28, wherein thenutritional supplement is formulated as a capsule, a pill, or asublingual tablet.
 30. The composition of claim 1, wherein theimmunostimulatory nucleic acid is formulated for local administration.31. The composition of claim 1, wherein the immunostimulatory nucleicacid is formulated for parenteral administration.
 32. The composition ofclaim 1, wherein the immunostimulatory nucleic acid is formulated in asustained release device.
 33. The composition of claim 1, wherein theimmunostimulatory nucleic acid is formulated for delivery to a mucosalsurface.
 34. The composition of claim 1, wherein the mucosal surface isselected from the group consisting of an oral, nasal, rectal, vaginal,and ocular surface.
 35. The composition of claim 1, wherein theimmunostimulatory nucleic acid stimulates a mucosal immune response. 36.The composition of claim 1, wherein the immunostimulatory nucleic acidstimulates a systemic immune response.
 37. The composition of claim 1,wherein the immunostimulatory nucleic acid is provided in an amounteffective to stimulate a mucosal immune response.
 38. The composition ofclaim 1, wherein the immunostimulatory nucleic acid is provided in anamount effective to stimulate a systemic immune response.
 39. Thecomposition of claim 1, wherein the immunostimulatory nucleic acid isprovided in an amount effective to stimulate an innate immune response.40. The composition of claim 1, wherein the immunostimulatory nucleicacid is provided in an amount effective to treat or prevent aninfectious disease.
 41. The composition of claim 1, wherein theimmunostimulatory nucleic acid is provided in an amount effective totreat or prevent an allergy.
 42. The composition of claim 1, wherein theimmunostimulatory nucleic acid is provided in an amount effective totreat or prevent asthma.
 43. The composition of claim 1, wherein theimmunostimulatory nucleic acid is provided in an amount effective totreat or prevent a cancer.
 44. The composition of claim 32, wherein thesustained release device is a microparticle.
 45. The composition ofclaim 40, wherein the infectious disease is a herpes simplex virusinfection.
 46. A method for stimulating an immune response in a subjectin need thereof comprising administering to a subject animmunostimulatory nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:1, in an amount effective to stimulate an immuneresponse.
 47. The method of claim 46, wherein the subject has or is atrisk of developing an infection.
 48. The method of claim 47, wherein theinfection is selected from the group consisting of a bacterialinfection, a viral infection, a fungal infection, and a parasiteinfection.
 49. The method of claim 48, wherein the viral infection isselected from the group consisting of Human immunodeficiency viruses(HIV-1 and HIV-2), Human T lymphotropic virus type I (HTLV-I), Human Tlymphotrophic virus type II (HTLV-II), Herpes simplex virus type I(HSV-1) Herpes simplex virus type 2 (HSV-2), Human papilloma virus(multiple types), Hepatitis A virus, Hepatitis B virus, Hepatitis C andD viruses, Epstein-Barr virus (EBV), Cytomegalovirus and Molluscumcontagiosum virus.
 50. The method of claim 49, wherein the viralinfection is a herpes simplex virus infection.
 51. The method of claim46, wherein the subject has or is at risk of developing allergy.
 52. Themethod of claim 46, wherein the subject has or is at risk of developingasthma.
 53. The method of claim 46, wherein the subject has or is atrisk of developing a cancer.
 54. The method of claim 46, furthercomprising administering an antigen to the subject.
 55. The method ofclaim 53, wherein the antigen is selected from the group consisting of amicrobial antigen, a cancer antigen, a self antigen, and an allergen.56. The method of claim 54, wherein the microbial antigen is selectedfrom the group consisting of a bacterial antigen, a viral antigen, afungal antigen, and a parasitic antigen.
 57. The method of claim 55,wherein the antigen is derived from a microorganism selected from thegroup consisting of herpesviridae, retroviridae, orthomyroviridae,toxoplasma, haemophilus, campylobacter, clostridium, E.coli, andstaphylococcus.
 58. The method of claim 46, wherein the immune responseis an antigen-specific immune response.
 59. The method of claim 53,wherein the antigen is encoded by a nucleic acid vector.
 60. The methodof claim 59, wherein the nucleic acid vector is separate from theimmunostimulatory nucleic acid.
 61. The method of claim 54, wherein theantigen is a peptide antigen.
 62. The method of claim 46, furthercomprising administering an adjuvant to the subject.
 63. The method ofclaim 62, wherein the adjuvant is a mucosal adjuvant.
 64. The method ofclaim 46, further comprising administering to the subject a secondtherapeutic agent.
 65. The method of claim 64, wherein the secondtherapeutic agent is an anti-microbial agent.
 66. The method of claim65, wherein the anti-microbial agent is selected from the groupconsisting of an anti-bacterial agent, an anti-viral agent, ananti-fungal agent, and an anti-parasite agent.
 67. The method of claim64, wherein the second therapeutic agent is an anti-cancer agent. 68.The method of claim 67, wherein the anti-cancer agent is selected fromthe group consisting of a chemotherapeutic agent, a cancer vaccine, andan immunomodulatory agent.
 69. The method of claim 64, wherein thesecond therapeutic agent is an allergy/asthma medicament.
 70. The methodof claim 69, wherein the allergy/asthma medicament is selected from thegroup consisting of PDE-4 inhibitor, bronchodilator/beta-2 agonist,K+channel opener, VLA-4 antagonist, neurokin antagonist, TXA2 synthesisinhibitor, xanthanine, arachidonic acid antagonist, 5 lipoxygenaseinhibitor, thromboxin A2 receptor antagonist, thromboxane A2 antagonist,inhibitor of 5-lipox activation protein, and protease inhibitor.
 71. Themethod of claim 46, wherein the immunostimulatory nucleic acid has anucleotide backbone which includes at least one backbone modification.72. The method of claim 71, wherein the backbone modification is aphosphorothioate modification.
 73. The method of claim 71, wherein thenucleotide backbone is chimeric.
 74. The method of claim 71, wherein thenucleotide backbone is entirely modified.
 75. The method of claim 46,wherein the immunostimulatory nucleic acid is free of methylated CpGdinucleotides.
 76. The method of claim 46, wherein the immunostimulatorynucleic acid includes a poly-G sequence.
 77. The method of claim 46,wherein the immunostimulatory nucleic acid is administered orally. 78.The method of claim 46, wherein the immunostimulatory nucleic acid isadministered locally.
 79. The method of claim 46, wherein theimmunostimulatory nucleic acid is administered parenterally.
 80. Themethod of claim 46, wherein the immunostimulatory nucleic acid isadministered in a sustained release device.
 81. The method of claim 46,wherein the immunostimulatory nucleic acid is administered to a mucosalsurface.
 82. The method of claim 46, wherein the immune response is amucosal immune response.
 83. The method of claim 46, wherein the immuneresponse is a systemic immune response.
 84. The method of claim 81,wherein the mucosal surface is selected from the group consisting of anoral, nasal, rectal, vaginal, and ocular surface.
 85. The method ofclaim 46, further comprising isolating an immune cell from the subject,contacting the immune cell with an effective amount to activate theimmune cell of the immunostimulatory nucleic acid and re-administeringthe activated immune cell to the subject.
 86. The method of claim 85,wherein the immune cell is a leukocyte.
 87. The method of claim 85,wherein the immune cell is a dendritic cell.
 88. The method of claim 85,further comprising contacting the immune cell with an antigen.
 89. Themethod of claim 46, wherein the subject is a human.
 90. The method ofclaim 46, wherein the subject is selected from the group consisting of adog, cat, horse, cow, pig, sheep, goat, chicken, monkey and fish. 91.The method of claim 46, wherein the subject has or is at risk ofdeveloping an infectious disease and wherein the method is a method fortreating or preventing the infectious disease.
 92. The method of claim46, wherein the subject has or is at risk of developing asthma and themethod is a method of treating or preventing asthma in the subject. 93.The method of claim 46, wherein the subject has or is at risk ofdeveloping allergy and the method is a method of treating or preventingallergy.
 94. The method of claim 46, wherein the subject has or is atrisk of developing a cancer and the method is a method of treating orpreventing the cancer.
 95. The method of claim 94, wherein the cancer isselected from the group consisting of biliary tract cancer; bone cancer;brain and CNS cancer; breast cancer; cervical cancer; choriocarcinoma;colon cancer; connective tissue cancer; endometrial cancer; esophagealcancer; eye cancer; gastric cancer; Hodgkin's lymphoma; intraepithelialneoplasms; larynx cancer; lymphomas; liver cancer; lung cancer (e.g.small cell and non-small cell); melanoma; neuroblastomas; oral cavitycancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer;sarcomas; skin cancer; testicular cancer; thyroid cancer; and renalcancer.
 96. The method of claim 46, further comprising administering anantibody specific for a cell surface antigen, and wherein the immuneresponse results in antigen dependent cellular cytotoxicity (ADCC). 97.A method for preventing disease in a subject, comprising administeringto the subject an immunostimulatory nucleic acid on a regular basis toprevent disease in the subject, wherein the immunostimulatory nucleicacid has a nucleotide sequence comprising SEQ ID NO:1.
 98. A method forinducing an innate immune response, comprising administering to thesubject an immunostimulatory nucleic acid in an amount effective foractivating an innate immune response, wherein the immunostimulatorynucleic acid has a nucleotide sequence comprising SEQ ID NO:1.
 99. Amethod for identifying an immunostimulatory nucleic acid comprisingmeasuring a control level of activation of an immune cell populationcontacted with an immunostimulatory nucleic acid comprising a nucleotidesequence of SEQ ID NO:1, measuring a test level of activation of animmune cell population contacted with a test nucleic acid, and comparingthe control level of activation to the test level of activation, whereina test level that is equal to or above the control level is indicativeof an immunostimulatory nucleic acid.